2011

Annual Report

BSRG | Biological Sciences Research Group

20 11 Annual Report

Contents

4 Executive Summary

5 Main Indicators

6 Research Programs Highlights

39 Publications

46 Communications in International Conferences

48 Communications in National Conferences

50 Other Scientific Activities

51 BSRG Members

Executive Summary

Molecular Systems and Synthetic Biology approaches were

extensively explored in the Biological Sciences Research

Group (BSRG) during 2011, focusing on the response and

resistance to environmental challenges. Research strategies

based on Omics analyses, including transcriptomics, prote-

omics, chemogenomics, metabolomics and lipidomics, bioinfor-

matics and more in-depth studies were used to obtain an inte-

grative perspective of key scientific questions in Biotechnology

and Biomedicine. In the field of Yeast Toxicogenomics we set

out to obtain mechanistic insights and a genome-wide view on

the responses to chemical compounds relevant in Environmen-

tal Health, Pharmacology and Biotechnology, to characterize

new signalling pathways, to model and understand gene regu-

latory networks under chemical stress and to identify molecular

biomarkers of drug/toxicant exposure. Promising yeast putative

efflux pumps were identified and the encoding genes were

successfully used in the design and construction of plants re-

sistant to agricultural relevant stresses. Bacterial Patho-

genomics studies led to mechanistic insights into adaptive

mechanisms of Burkholderia cepacia complex bacteria to the

stressing environment of the lung of cystic fibrosis patients

under antimicrobial therapy during long-term infection. In col-

laboration with computer biologists, our activity also involved

the update of the information on yeast transcriptional regulato-

ry associations available in the YEASTRACT database and the

programmatic access to this curated information, the applica-

tion of a new ranking algorithm to our genome-wide transcrip-

tion data to prioritize regulatory associations, and the modelling

of a gene regulatory network. Genome-wide identification of

candidate genes and signalling pathways to guide yeast ro-

bustness engineering to improve bioethanol production and

food preservation strategies was another objective of our re-

search envisaging a synthetic biology approach. In collabora-

tion with BERG, a quantitative proteomic analysis unveiled the

expression program of human mesenchymal stem cells upon

extended ex-vivo cultivation.

Other achievements in the field of Molecular and Cellular Mi-

crobiology include: the identification and molecular characteri-

zation of B. cenocepacia virulence factors and genes/proteins

with potential impact in diagnostic and design of new antimicro-

bials against this bacterial pathogen, in particular trimeric auto-

transporter adhesins and the second RNA chaperone encoding

gene hfq2; the potential therapeutic application of the bacterial

protein azurin and its truncated peptides as new anticancer

agents; the functional analysis of S. cerevisiae drug efflux

pumps and of their role in cell defence and the extrapolation of

this knowledge to the functional analysis of homologous solute

transporters in the plant model Arabidopsis thaliana and in the

opportunistic pathogenic yeast Candida glabrata.

The research activities of the BSRG during 2011 led to the

publication of 26 articles in international peer-reviewed jour-

nals, 5 of them invited review-articles, 4 book chapters and the

co-edition of a special issue of the International Journal of Mi-

crobiology, and delivered 4 PhD and 6 MSc theses and 2 pa-

tent applications. New promising activities were initiated or

consolidated based on our participation in national projects and

in projects in the frame of ERA-NETs Pathogenomics and In-

dustrial Biotechnology and of a COST Action. The reasonable

level of competitive funding obtained in 2011 for research pro-

jects and PhD and post-doctoral fellowships enabled the devel-

opment of a critical mass of sustainable research capacity and

the further consolidation of an internationally recognized re-

search group, especially in emerging areas of Biological Sci-

ences.

Isabel Sá-Correia

Head of the BSRG

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Executive Summary

Human Resources

In 2011, the BSRG had 54 members ( 40 Females and 14 Males), including 13 PhD holders.

Publications

The output of BSRG activities published from January 1 to December 31 2011 includes 26 articles in peer-reviewed internati-

onal journals, 4 book chapters, the edition of 1 book and of 1 special issue of an international journal, and 2 patent applicati-

ons.

Main Indicators

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Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Sandra dos Santos, Margarida Palma,

Paulo J. Dias

PhD and MSc students: Artur B. Lourenço, Tânia, R. Cabrito, Andreia Madeira, Catarina Rodrigues, Ana S. Moreira, Joana F. Guerreiro, Filipa C. Roque, Rita Maldonado, Sílvia Henriques, Kaur Alasoo

Molecular Systems Microbiology

Objectives

A Molecular Systems Microbiology approach, based

on the integration of Omics analyses, including tran-

scriptomics, expression proteomics and phosphopro-

teomics, chemogenomics, metabolomics and lip-

idomics, with bioinformatics was explored during

2011. This systems level biological information was

used to get an integrative view on how microbial cells

with important roles in biotechnology, human health,

agriculture and the environment, function and re-

spond to drugs and other environmental stresses.

Although more focused on the model eukaryote and

industrial yeast species Saccharomyces cerevisiae

this approach was successfully extended to

Burkholderia cepacia complex bacteria.

Gene and genomic regulation in response to environ-

mental challenges were examined focusing on the

underlying mechanisms and signalling pathways, and

on the characterization and modelling of gene regula-

tory networks.

Research Activities

1. Yeast toxicogenomics

Mechanistic insights and a genome-wide view on the

responses to chemical compounds relevant in Envi-

ronmental Heath [9], Pharmacology [3] and Biotech-

nology [5], were gathered to characterize new signal-

ling pathways, to model and understand gene regula-

tory networks under stress and to identify molecular

biomarkers of drug/toxicant exposure (further de-

scribed in pages 8 and 22 highlights).

Genome-wide identification of candidate genes and

signaling pathways for yeast robustness engineering

to improve bioethanol production [6, 10] and food

preservation strategies [5, 6, 11] was another objec-

tive of our research using the synthetic biology ap-

proach (further described in pages 8 and 22 high-

lights).

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2. Gene and genomic regulation

Understanding how different transcription regulato-

ry networks overlap and cross-talk to influence tran-

scription in yeast under optimal conditions of

growth or under environmental stress is the aim of

our research activities.

In collaboration with computer biologists, our activi-

ty also involved the update of yeast transcription

regulation associations available in the

YEASTRACT database [1] and the upgrade of this

information system and the modelling of gene regu-

latory networks [4,9] (further described in page 12

highlight).

3. Bacterial Pathogenomics

Pathogenomics´ studies involved the elucidation of

adaptation mechanisms of Burkholderia cepacia

complex bacteria to the stressing environment of

the lung of cystic fibrosis patients and antimicrobial

therapy during long-term colonization [2, 7, 8]

(further described in page 29).

Selected Publications

[1] Abdulrehman, D., Monteiro, P.T., Teixeira,

M.C., Mira, N.P., Lourenço, A.B., dos Santos,

S.C., Cabrito, T.R., Francisco, A., Madeira, S.C.,

Santos, R., Oliveira, A.L., Sá-Correia I., Freitas,

A. T., “YEASTRACT: Providing a programmatic

access to curated transcriptional regulatory associ-

ations in Saccharomyces cerevisiae through a web

services interface”, Nucleic Acids Research, 39:

D136-140, 2011.

[2] Coutinho, C.P., dos Santos, S.C., Madeira,

A., Mira, N.P., Moreira, A.S., Sá-Correia, I., Long-

term colonization of the cystic fibrosis lung by

Burkholderia cepacia complex bacteria: epidemiolo-

gy, clonal variation and genome-wide expression

alterations. Frontiers in Cellular and Infection

Microbiology, 1:12, 2011.

[3] dos Santos, S.C., Sá-Correia, I., A genome-

wide screen identifies yeast genes required for pro-

tection against or enhanced cytotoxicity of the anti-

malarial drug quinine. Molecular Genetics and

Genomics, 286: 333-346, 2011.

[4] Gonçalves, J.P., Francisco, A.P., Mira, N.P.,

Teixeira, M.C., Sá-Correia, I., Oliveira, A.L., Ma-

deira, S.C., TFRank: Network-based prioritization

of regulatory associations underlying transcriptional

responses, Bioinformatics, 27, 3149-3157, 2011.

[5] Lourenço, A.B., Ascenso, J.R., Sá-Correia, I.,

“Metabolic insights into the yeast response to propi-

onic acid based on high resolution 1H-NMR spec-

troscopy”, Metabolomics, 7, 457-468, 2011.

[6] Mira, N.P., Henriques, S., Keller, G., Teixeira,

M.C., Matos, R., Arraiano, C., Winge, D.R., Sá-

Correia, I., Identification of a DNA binding site for

the transcription factor Haa1p, required for Saccha-

romyces cerevisiae response to acetic acid stress.

Nucleic Acids Research, 16, 6896-907, 2011.

[7] Mira, N.P., Madeira, A., Moreira, A.S.,

Coutinho, A.P., Sá-Correia, I., Genomic expres-

sion analysis reveals strategies of Burkholderia

cenocepacia to adapt to the airways of cystic fibro-

sis patients and antimicrobial therapy . PLOS One,

6: e28831.

[8] Madeira, A., Santos, P.M., Coutinho, C.P.,

Pinto-de-Oliveira, A., Sá-Correia, I., Quantitative

proteomics (2-D-DIGE) reveals molecular strate-

gies employed by Burkholderia cenocepacia to

adapt to the airways of cystic fibrosis patients under

antimicrobial therapy. PROTEOMICS, 11, 1313-28.

[9] Monteiro P.T., Dias J.P., Ropers D., Oliveira

A.L., Sá-Correia I., Teixeira M.C., Freitas A.T.,

"Qualitative modeling and formal verification of the

FLR1 gene mancozeb response in Saccharomyces

cerevisiae", IET Systems Biology, 5, 308-316,

2011.

[10] Pereira, F.B., Guimarães, P.M.R., Gomes,

D.G., Mira, N.P., Teixeira, M.C., Sá-Correia, I.,

Domingues, L., Identification of candidate genes

for yeast engineering to improve bioethanol produc-

tion in Very-High-Gravity and lignocellulosic bio-

mass industrial fermentations. Biotechnology for

Biofuels, 4, 57, 2011.

[11] Teixeira, M.C., Mira, N.P., Sá-Correia, I., “A

genome-wide perspective on the response and

tolerance to food-relevant stresses in Saccharomy-

ces cerevisiae”, Current Opinion in Biotechnolo-

gy, 22:150-156, 2011.

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Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Sandra C. dos Santos, Paulo J. Dias

PhD and MSc students: Andreia Madeira, Catarina R. Rodrigues, Artur B. Lourenço, Tânia R. Cabrito, Joana

F. Guerreiro, Filipa C. Roque, Ana S. Moreira, Rita Maldonado

Objectives

Proteomics and metabolomics offer the possibility to

gain global insights at two different molecular levels

in a biological system. Proteomics, and in particular 2

-dimensional electrophoresis (2-DE)-based quantita-

tive proteomics, allows the study of proteome-wide

alterations occurring in a cell at the level of expres-

sion, localisation and post-translational modifications

in response to genomic or environmental changes

(Sá-Correia I., Teixeira M.C., Expert Rev Proteomics,

7: 943-953, 2010). Metabolomics, and specifically

NMR-based metabolomics, enables the non-biased

identification of metabolome-wide alterations occur-

ring in a cell. Proteomics and Metabolomics, either

as stand-alone or as complementary approaches,

are crucial to gain a full understanding of the dynam-

ic and complex behaviour of biological systems in

response to genomic or environmental changes.

In this context, our research group explored the ex-

pression proteomics and/or the NMR-based metabo-

lomics platforms during 2011 with the following ob-

jectives:

1. Investigate at the metabolome level the effect

of the food preservative propionic acid and of the

fermentation product ethanol in Saccharomyces

cerevisiae.

2. Study the mechanisms of persistence and

adaptive evolution of Burkholderia cenopacia bacte-

ria in the lungs of cystic fibrosis (CF) patients.

3. Characterise the proteome-wide responses,

including alterations at the phospho-proteome level,

induced by the anticancer drug imatinib in S. cere-

visiae cells, and by acetic acid in the food spoilage

yeast Zygosaccharomyces bailii.

4. Characterize the proteome-wide responses in

Pseudomonas sp. M1 cells grown on -myrcene to

engineer its metabolic pathways

5. Describe the proteome alterations underlying

extended ex-vivo culture of human mesenchymal

stem cells (MSC), in order to contribute to optimisa-

tion and quality control of MSC expansion for thera-

peutic use.

Research Topics

1. Yeast metabolomics in food and biotechnology

research

Economic impact is one the major factors to consider

in any industrial process. Studies focusing on bioeth-

anol production and food preservation strategies

have receiving a great deal of attention due to their

economic impact in different biotechnology indus-

tries. S. cerevisiae has been widely used as a model

to elucidate the molecular mechanisms behind re-

sistance to weak acid food preservatives in fungi and

to expand the knowledge behind the molecular

mechanisms of resistance to the multiples stresses

found during alcoholic fermentation (see page 6).

Metabolomics, the less explored global-approach, is

becoming increasingly popular, since it can be re-

garded as the end-point of the omics cascade and

the metabolome as a chemical fingerprint of the or-

ganism. Using a NMR-based metabolomics ap-

proach, the time-dependent effect of the food pre-

servative propionic acid in the yeast metabolome

was examined [1]. This work will be extended to oth-

er weak acids, namely acetic acid, aiming the identifi-

cation of similarities and differences in their mode of

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in biological research

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action. A similar metabolomics approach was used to

gain insights into the yeast response to ethanol, where

the effect of increasing ethanol concentrations and the

effect of FPS1 expression on the metabolome were

analysed. This analysis is being extended to other

specific genes.

2. Proteomics applied to food preservation

Z. bailii is considered the most tolerant yeast species

to acetic acid-induced toxicity, being able to grow in

the presence of concentrations of this food preserva-

tive close to the legal limits. For this reason, Z. bailii is

the most important microbial contaminant of acidic

food products but the mechanisms behind this intrinsic

resistance to acetic acid are very poorly characterised.

To gain insights into the adaptive response and toler-

ance to acetic acid in Z. bailii, we explored an expres-

sion proteomics approach, based on quantitative 2-DE,

to identify alterations occurring in the protein content in

response to sudden exposure or balanced growth in

the presence of an inhibitory but non-lethal concentra-

tion of this weak acid [2]. This analysis is being extend-

ed to lethal acetic acid concentrations.

3. Proteomics applied to -myrcene pathway engi-

neering

Identify proteins whose content is altered in Pseudo-

monas sp. M1 cells when cultivated in -myrcene, to

understand the molecular adaptation mechanisms and

to guide the engineering of pathways to produce -

myrcene derivatives with potential application in cos-

metic and pharmaceutical industry.

4. Stem cell Proteomics

Human mesenchymal stem cells (MSC) have been on

the focus of intense clinical-oriented research due to

their multilineage differentiation potential and immuno-

modulatory properties. However, to reach the clinically

meaningful cell numbers for cellular therapy and tissue

engineering applications, MSC ex-vivo expansion is

mandatory but sequential cell passaging results in loss

of proliferative, clonogenic and differentiation potential.

To get clues into the molecular mechanisms underly-

ing cellular senescence resulting from extended ex-

vivo cultivation of bone marrow MSC, we explored 2-

DE-based quantitative proteomics to compare the ex-

pression programs of Passage 3 cells, commonly used

in clinical studies with expanded MSC, and Passage 7

cells, which already demonstrated significant signs of

culture-induced senescence [3].

The final goal of this research program, carried out in

collaboration with the BERG group, IBB/CEBQ, IST, is

to understand how the ex-vivo culture process affects

MSC expansion at the proteome level. This genome-

wide expression approach has already proven useful

for revealing the molecular mechanisms underlying the

decrease of proliferative and clonogenic potential of P7

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vs. P3 cells, and paves the way for setting up a pro-

teome profiling strategy for quality control to ensure

safe and clinically effective expanded MSC.

5. Pathoproteomics and Pathometabolomics in Bcc

bacteria clinical isolates

Chronic respiratory infections caused by B. cenoce-

pacia in CF patients are characterised by low re-

sponsiveness to antibiotic therapy and, in general,

to a more rapid decline of lung function (see page

29). To obtain clues into the molecular mechanisms

underlying the adaptive strategies employed to deal

with the stressing conditions of the CF lung, quanti-

tative proteomics (2-D fluorescence Difference Gel

Electrophoresis (DIGE) analysis combined with

MALDI-TOF MS/MS) was used to compare the ex-

pression programs of pairs of clonal isolates re-

trieved from a chronically infected CF patient [ref. 4

and unpublished results].

Comparative analyses of the exoproteomes and

exometabolomes of different Bcc clinical isolates

can be helpful to gain novel insights into the contri-

bution of exported proteins and metabolites to Bcc

virulence in a CF context. As such, we are currently

applying exoproteomics and exometabolomics ap-

proaches to four sequential B. cenocepacia clonal

variants, using a DIGE analysis combined with

MALDI-TOF MS/MS and the two main analytical

platforms in the field of metabolomics, one based on

mass spectrometry and one based on nuclear mag-

netic resonance spectroscopy. We are also plan-

ning to extend the exoproteomes and exometabo-

lomes analyses to clinical variants of other Bcc spe-

cies obtained from different CF patients (e.g. B.

dolosa and B. cepacia species).

Main Achievements

The metabolomic analysis, based on 1H-NMR

spectroscopy, highlighted the separation of the

metabolomes of cells harvested during propionic

acid-induced lag-phase in two parts, correlating with

cell population viability and average intracellular pH

(pHi) profiles [1]. Among other metabolic changes,

an association between the average pHi values and

the levels of glutamate and propionate during

growth latency was identified.

Characterisation of the S. cerevisiae metabo-

lome in the presence of increasing ethanol concen-

trations (unpublished results). The metabolomic

analysis confirmed the role of Fps1p in glycerol ho-

meostasis and revealed a marked dose-dependent

accumulation of trehalose, an increased pool of a

number of amino acids and an alteration in redox

state in response to ethanol.

Design of a quantitative- and phospho-

proteomics study applied to pharmacogenomics to

monitor the response of yeast cells to an inhibitory

concentration of the anticancer drug imatinib, with

main objective of identifying new signalling path-

ways potentially involved in the response to this

drug. The results highlighted the importance of glu-

coneogenesis and glycolytic pathways, which have

recently been associated with the mechanism of

imatinib action in human cell lines (submitted re-

sults).

Identification of 79 proteins differentially ex-

pressed between two sequential B. cenocepacia

isolates (IST439 and IST4113, see pages 29),

mainly involved in carbohydrate metabolism, trans-

lation, iron uptake, nucleotide synthesis, protein

folding, peptidoglycan, membrane lipids and lipopol-

ysaccharide synthesis [2]. A similar approach was

adopted for the comparison of IST439 and IST4134,

and the results are currently being integrated

(unpublished results). The quantitative comparisons

of the different proteomes suggests a genetic adap-

tation leading to increased bacterial persistence in

the CF airways.

Identification of 79 protein forms whose content

is altered in P7 vs P3 MSC cells. The large number

of multiple isoforms with an altered content identi-

fied in P7 vs P3, namely the cytoskeleton compo-

nents β-actin (7 forms) and vimentin (24 forms),

emphasises the importance of post-transcriptional

modification upon long-term cultivation (under revi-

sion in PLoS One). The differential protein expres-

sion registered suggests that cellular senescence

occurring during ex-vivo expansion of MSC is asso-

ciated with the impairment of cytoskeleton remodel-

ling and/or organisation and the repair of damaged

proteins.

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Identification of Z. bailii cells response at the pro-

teome level to the supplementation of a glucose-

containing growth medium with an inhibitory but sub-

lethal concentration of acetic acid. Results suggest a

cellular metabolic reprogramming where glucose and

acetic acid are co-consumed and acetate is channeled

into the tricarboxylic acid cycle (under revision in Pro-

teomics).

Insights into the alterations induced by β-myrcene

on the membrane proteome of Pseudomonas sp. M1

cells were obtained based on an expression prote-

omics analysis carried out using liquid chromatography

coupled with mass spectrometry (LC-MS) after label-

ling with isobaric tags for relative and absolute quanti-

tation (iTRAQ). The results are currently under analy-

sis.

Funded Projects

ZygoSacAR - Mechanistic insights into acetic acid

resistance in food spoilage yeasts: from the experi-

mental Saccharomyces cerevisiae to Zygosaccharo-

myces spp., PTDC/AGR-ALI/102608/2008, PI: Isabel

Sá-Correia.

ADHRES - Signature Project: Expression profiling

of adhesive (AHD) and resistance (RES) genes in bio-

film lifestyle in P. aeruginosa, P. putida and B. cenoce-

pacia, in the frame of the ERA-NET Pathogenomics,

ERA-PTG/SAU/0001/2008, PI: Isabel Sá-Correia.

Imatinib resistance and targets in chronic myeloid

leukaemia: post-genomic approaches using yeast as a

model system, PTDC/SAU-FCF/71760/2006, PI: Isabel

Sá-Correia.

Towards the metabolic engineering of beta-

myrcene pathway of Pseudomonas sp. M1: functional

genomics and structural biochemistry approaches,

PTDC/EBB-BIO/104980/2008, PI at IST: Isabel Sá-

Correia.

IBB – LA Project – Strategic Area PEst- OE/EQB/

LA0023/2011_research lines: Systems and Synthetic

Biology and Stem Cell Engineering and Regenerative

Medicine.

Selected Publications

[1] Lourenço, A.B., Ascenso, J.R., and Sá-Correia,

I., “Metabolic insights into the yeast response to propi-

onic acid based on high resolution 1H-NMR spectros-

copy”, Metabolomics 7: 457-468, 2011.

[2] Madeira, A., Santos, P.M., Coutinho, C.P., Pinto-

de-Oliveira, A., and Sa-Correia, I., “Quantitative pro-

teomics (2-D DIGE) reveals molecular strategies em-

ployed by Burkholderia cenocepacia to adapt to the

airways of cystic fibrosis patients under antimicrobial

therapy”, Proteomics 11: 1313-1328, 2011.

12

Gene and genomic regulation: From Mecha-

nisms and signalling pathways to Gene

Regulatory Networks

Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Paulo J. Dias, Sandra C. dos Santos

PhD and MSc students: Artur B. Lourenço, Tânia R. Cabrito, Sílvia Henriques, Cláudia Godinho, Kaur Alasoo

Objectives

Transcriptional regulation depends on the action of

transcription factors (TFs), activators and/or re-

pressors, that bind to specific DNA sequences pre-

sent in the promoter region of target genes and

thereby modulate transcription. At a global scale, the

transcript level of a given gene results from the con-

certed action of these specific TFs, operating in inter-

twined and complex regulatory networks. In the field

of gene and genomic expression the main objectives

of our research aim at:

1. Understanding how different transcription

regulatory networks overlap and cross-talk to influ-

ence transcription in yeast under optimal conditions

of growth or under environmental stress;

2. Elucidating the molecular mechanisms by

which the promoter context and environmental

conditions modulate the binding and action of tran-

scription factors;

3. Exploring and developing computational tools

to build suitable predictive models of transcriptional

regulatory networks active in yeast, and to extrapo-

late them to less genetically accessible eukaryotes.

Research Topics

1. Global analysis of the regulators controlling the

adaptive response and resistance to

weak acid stress in yeasts

Two transcriptional regulatory networks, dependent

on the transcription factors Haa1p (Fernandes et al.,

2005, Biochem Biophys Res Commun, 337: 95-103)

and Rim101p (Mira et al., 2009, FEMS Yeast Re-

search, 9, 202-216), were recently implicated by our

research group in yeast response and resistance to

acetic and propionic acids, based on transcriptomic

and chemogenomics approaches. Resistance of

spoilage yeasts and fungi to these food preservative

weak acids is a major concern. The role played by

the Haa1p signalling pathway in S. cerevisiae re-

sponse and resistance to these acids is being exam-

ined at the proteome and metabolome level. The

identification of a Haa1p-like regulator and signalling

pathway in the spoilage yeast Zygosaccharomyces

bailii, intrinsically more tolerant to weak acids than S.

cerevisiae, is envisaged. Expression proteomics and

metabolomics are also being used to elucidate the

adaptive response to acetic acid stress in Z. bailli

and the role of the Haa1p-like protein in that global

response.

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2. Understanding transcription factor-DNA interac-

tions based on structural- and nanobiotechnology-

based approaches

The transcription factor Haa1 is the main player in

reprogramming yeast genomic expression in re-

sponse to acetic acid stress. Microarray analysis

was used to obtain the list of genes whose expres-

sion changes in the dependency of Haa1 in re-

sponse to acetic acid stress (Mira et al., 2010, OM-

ICS, 14: 587-601). Surface Plasmon Resonance

and electrophoretic mobility shift assays were used

to identify Haa1 binding site. The Haa1-dependent

transcriptional regulatory network active in the yeast

response to acetic acid stress is being re-evaluated

based on the knowledge of Haa1 direct target

genes. The development of a nanostructured acous-

tic wave biosensor for the detection and analysis of

transcription factor interactions is ongoing, in collab-

oration with IBB/CBME, University of Algarve.

3. Development and validation of bioinformatics

tools to describe complex transcription regulatory

networks

Computational tools are being applied to the

management and analysis of the massive amount of

data emerging from genome-wide expression

analyses, in collaboration with the KDBIO Group of

INESC-ID, envisioning the delineation of the com-

plex networks underlying yeast responses to chemi-

cals of biomedical and biotechnological relevance.

Joint activities in the field of Bioinformatics applied

to the understanding of global transcriptional regula-

tory networks involved the creation (Teixeira et al.,

2006, Nucleic Acids Res. 34: D446-D451) and

maintenance, updating and upgrading of the

YEASTRACT database (Monteiro et al., 2008, Nu-

cleic Acids Res. 36: D132-D136), as well as the

development and application to biological analysis

of a new ranking algorithm, TFRank, better suited

than previous tools for network-based prioritization

of regulatory associations predicted to underlie a

given genome-wide expression response.

The development and test of algorithms to model

complex transcription regulatory networks, such as

that underlying the transcriptional activation, under

mancozeb stress, of the multidrug resistance trans-

porter encoded by the FLR1 gene (Teixeira et al.,

2010, Mol Biosyst, 6: 2471-2481) are also among

the joint research activities.

Main Achievements

A new version of the YEASTRACT database

was released. While the first articles describing the

database were cited over 200 times (according to

ISI web of Knowledge) since 2006, a new one was

published in the 2011 Nucleic Acids Research Data-

base Issue [1].

A new ranking algorithm developed at INESC-ID

was validated to prioritize the transcription factors

predicted to underlie genome-wide expression

changes [2].

A minimal functional binding site of Haa1 was

identified - 5’-(G/C)(A/C)GG(G/C)G-3’ - allowing the

prediction of the direct targets of Haa1, up-regulated

in response to acetic acid stress [3].

The complex transcriptional regulatory network

underlying the activation of FLR1 gene expression

was modelled using qualitative modelling and formal

verification techniques. The simulated behaviour of

the network was verified experimentally, shedding

new light into its characteristics and functioning [4].

The combinatorial transcription regulation of

TPO1 involving the transcriptional factors Pdr1,

Pdr3, Gcn4, Stp1 and Stp2 in yeast cells exposed to

benzoic acid is being analyzed, aiming the modeling

of this regulatory network, in collaboration with the

Network Modelling Group, IGC.

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Funded Projects

ZygoSacAR - Mechanistic insights into acetic acid

resistance in food spoilage yeasts: from the experi-

mental model Saccharomyces cerevisiae to Zygo-

saccharomyces spp, PTDC/AGR-ALI/102608/2008,

PI: Isabel Sá-Correia.

QCMTF-Biosensors for the analysis and detec-

tion of transcription factors, PTDC/EBB-

EBI/108517/2008, PI at IST: Isabel Sá-Correia.

Selected Publications

[1] Abdulrehman, D., Monteiro, P.T., Teixeira,

M.C., Mira, N.P., Lourenço, A.B., dos Santos,

S.C., Cabrito, T.R., Francisco, A., Madeira, S.C.,

Santos, R., Oliveira, A.L., Sá-Correia I., Freitas,

A. T., “YEASTRACT: Providing a programmatic

access to curated transcriptional regulatory associ-

ations in Saccharomyces cerevisiae through a web

services interface”, Nucleic Acids Research, 39:

D136-140, 2011.

[2] Gonçalves, J.P., Francisco, A.P., Mira, N.P.,

Teixeira, M.C., Sá-Correia, I., Oliveira, A.L., Ma-

deira, S.C., TFRank: Network-based prioritization

of regulatory associations underlying transcriptional

responses, Bioinformatics, 27, 3149-3157, 2011.

[3] Mira, N.P., Henriques, S., Keller, G., Teixeira,

M.C., Matos, R., Arraiano, C., Winge, D.R., Sá-

Correia, I., Identification of a DNA binding site for

the transcription factor Haa1p, required for Saccha-

romyces cerevisiae response to acetic acid stress.

Nucleic Acids Research, 16, 6896-907, 2011.

[4] Monteiro P.T., Dias J.P., Ropers D., Oliveira

A.L., Sá-Correia I., Teixeira M.C., Freitas A.T.,

"Qualitative modeling and formal verification of the

FLR1 gene mancozeb response in Saccharomyces

cerevisiae", IET Systems Biology, 5, 308-316,

2011.

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Drug efflux pumps in chemical stress defense

Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Paulo Dias

PhD and Msc students: Tânia R. Cabrito, Filipa Roque, Sílvia Henriques, Catarina Costa, Cláudia Godinho,

Kaur Alasoo, Joana Nunes, Carla Pires, André Henriques, Guida Camacho

Objectives

Multidrug resistance (MDR) is implicated in the failure

of many therapeutic, food-preservation and crop pro-

tection actions, but can also be explored in the im-

provement of biotechnological processes’ productivi-

ty. MDR is many times the result of the action of mul-

tidrug efflux transporters found in the plasma of all

living cells. In this context, our research aims to:

1. Carry out the functional analysis of MDR trans-

portes of the Major Facilitator Superfamily (MFS) and

of the ATP Binding Cassete (ABC) Superfamily in the

model eukaryote Saccharomyces cerevisiae, focus-

ing on their physiological role and their connection to

the MDR phenomenon.

2. Characterize the regulatory networks underly-

ing the transcriptional up-regulation of MDR trans-

porter encoding genes, using molecular biology and

computational approaches (see page 12).

3. Extend current knowledge on yeast MDR trans-

porters to the plant model Arabidopsis thaliana, ex-

ploring this information to engineer more tolerant

plants.

4. Extend current knowledge on S. cerevisiae

MDR transporters to fungal pathogens, particularly to

the pathogenic yeast Candida glabrata.

Research Topics

The emergence of multidrug resistance (MDR), i.e.

the simultaneous acquisition of resistance to a wide

range of structurally and functionally unrelated cyto-

toxic chemicals, is found in a wide variety of organ-

isms, from bacteria to mammals. This phenomenon

is many times dependent on the action of drug efflux

pumps that apparently mediate the expulsion of

drugs and other cytotoxic compounds from the cyto-

plasm to the outer medium, or the transport of so-

lutes across internal membranes leading to differen-

tial intracellular accumulation into the vacuole or oth-

er organelles, providing protection from their toxic

effects.

1. Functional analysis of S. cerevisiae MDR trans-

porters of the MFS

The yeast S. cerevisiae has a privileged position as a

tool to understand the mechanisms underlying the

action of cytotoxic insults in more complex and less

easily accessible eukaryotes. Since the release of

the yeast genome sequence, our research group has

been involved in the functional analysis of new MDR

transporters from the Major Facilitator Superfamily

(MFS) (Sá-Correia et al., 2009, Trends Microbiol, 17:

22-31) and from the ATP Binding Cassette (ABC)

superfamily. The ongoing research pursues a deeper

understanding of the physiological role of these drug

efflux pumps, their contribution to MDR, the complex

transcription networks involved in their regulation in

response to drugs/chemical stress, their evolution

and on how this knowledge can be extrapolated to

pathogenic fungi and higher eukaryotes.

Proposed model for polyamine homeostasis in S. cerevisiae

2. Extrapolation of the current knowledge on MDR

transporters to pathogenic yeasts and to the plant

model Arabidopsis thaliana.

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The use of S. cerevisiae as a model system to study

agricultural and clinical antifungal resistance has

provided promising clues to guide the study of anti-

fungal resistance in pathogenic and phytopathogenic

yeasts. In particular, this knowledge and experi-

mental approaches are being used to guide the func-

tional characterization of a family of MFS-MDR trans-

porters in the pathogenic yeast Candida glabrata.

The role of S. cerevisiae drug efflux pumps in re-

sistance to the herbicide 2,4-D has been character-

ized by our group over the last years (Teixeira et al.,

2007, Trends Biotech, 25: 363-370). The implications

of this knowledge in A. thaliana resistance to herbi-

cides and other pesticides and phytotoxics, and the

use of yeast as a heterologous expression system,

are being examined, in collaboration with the Plant

Molecular Biology (PMB) group, Instituto Gulbenkian

de Ciência (IGC). Several yeast and plant MFS

transporters are being characterized, focusing on

their physiological role and involvement in stress

tolerance (Cabrito et al., 2009, Appl Microbiol and

Biotechnol, 84: 927-936 and recently submitted re-

sults), as a result of this joint effort aiming the under-

standing of the biological role of this poorly charac-

terized family of proteins and the development of

stress-resistant crops.

Proposed model for ergosterol homeostasis in S. cerevisiae

Main Achievements

A physiological role was attributed to the MFS-

MDR transporter Qdr3 from S. cerevisiae, specifically

in the control of spermine and spermidine homeosta-

sis, under the regulation of the transcription factor

Yap1. These polyamines are essential organic poly-

cations that play multiple functions in the cell, includ-

ing the regulation of nucleic acid and protein synthe-

sis. An association between this physiological role

and Qdr3 participation in conferring resistance to

anticancer drugs is hypothesized [1].

The combinatorial transcription regulation of

FLR1 involving the transcriptional factors Yap1, Pdr3,

Yrr1 and Rpn4 in yeast cells exposed to the agricul-

tural fungicide mancozeb, was further examined

through a combination of computational and experi-

mental approaches and the qualitative modelling and

formal verification of the FLR1 gene mancozeb re-

sponse in Saccharomyces cerevisiae was possible

[2] (see pages 12 regulatory networks).

The combinatorial transcription regulation of

TPO1 involving the transcriptional factors Pdr1, Pdr3,

Gcn4, Stp1 and Stp2 in yeast cells exposed to ben-

zoic acid is being analyzed, aiming the modeling of

this regulatory network, in collaboration with the Net-

work Modelling Group, IGC (see pages 12 regulatory

networks).

Through a lipidomic analysis, a physiological role

was attributed to the ABC-MDR transporter Pdr18

from S. cerevisiae, specifically in plasma membrane

ergosterol incorporation. This physiological role af-

fects plasma membrane potential and is proposed to

underlie Pdr18 action as a MDR determinant, confer-

ring resistance to the herbicide 2,4-D, among other

xenobiotic compounds [3].

The response and resistance mechanisms to

herbicides in the model eukaryote S. cerevisiae and

the model plant A. thaliana, in particular those involv-

ing MDR transporters, including many of our recent

findings in yeast, were published as a book chapter

[4].

The inspection of the role of C. glabrata DHA1

family members indicated that the multidrug trans-

porter CgQdr1/2 confers imidazole antifungal drugs

resistance, playing a role in their efflux from C. gla-

brata cells.

The functional analysis of several plant MFS

transporters, with emphasis on their physiological

role and involvement in stress tolerance is progress-

ing. The strategies employed are based on those

successfully implemented with the plant ORF

At5g13750, which plays a role in 2,4-D resistance in

yeast (Cabrito et al., 2009, Appl Microbiol and Bio-

technol, 84: 927-936). The functional characterization

of two of these transporters, found to be involved in

phosphate and auxin homeostasis, was recently sub-

mitted for publication (Remy et al., New Phytol, under

minor revision; Remy et al., Plant Cell, under major

revision).

The yeast drug efflux pump Tpo1 was heterolo-

gously expressed in A. thaliana and the growth inhi-

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bition of plants exposed to several agricultural pesti-

cides was reduced (Patent Application – PT

105727, 28/10/2011). This technology was recently

selected to be presented in the technology exhibi-

tion AGROFOOD iTECH, at the “Salão Internacional

de Agro-Negócios” (SIAG) 2012.

Transcription regulatory network predicted to con-

trol TPO1 expression, based on the data gathered

in the YEASTRACT database

Funded Projects

Functional and biochemical analysis of the yeast

multidrug resistance transporters Qdr2 and Aqr1,

PTDC/BIA-MIC/72577/2006, PI: Miguel C. Teixeira.

Resistance to pesticides and other chemical

stresses of agricultural interest: Role of plant Major

Facilitator Superfamily Transporters, PTDC/AGR-

AAM/102967/2008, PI at IST: Isabel Sá-Correia.

FUNDRING: Identification of new biomarkers for

antiFUNgal Drug Resistance diagnosis IN Candida

Glabrata: the particular role of multidrug resistance

transporters, PTDC/EBB-BIO/119356/2010, PI: Mi-

guel C. Teixeira

INTACT: Integral Engineering of Acetic Acid Tol-

erance in Yeast, ERA-IB/0002/2010, PI: Isabel Sá-

Correia

Patents

Patent Application: PT 105727 - "Utilização de um

gene que confere resistência a xenobióticos em

plantas" Inventors: Sá-Correia, I., Cabrito, T.R.,

Teixeira, M.C., Remy, E., Duque, P. (28/10/2011)

Selected Publications

[1] Teixeira, M.C., Cabrito, T.R., Hanif, Z.M., Var-

gas, R.C., Tenreiro, S., Sá-Correia, I., “Yeast re-

sponse and tolerance to polyamine toxicity involving

the drug:H+ antiporter Qdr3 and the transcription

factors Yap1 and Gcn4”, Microbiology, 157: 945-

956, 2011.

[2] Monteiro, P.T., Dias, P.J., Ropers, D., Oliveira,

A.L., Sá-Correia, I., Teixeira, M.C., Freitas, A.T.,

“Qualitative modelling and formal verification of the

FLR1 gene mancozeb response in Saccharomyces

cerevisiae”, IET Systems Biology, 5(5): 308-16,

2011.

[3] Cabrito, T.R., Teixeira, M.C., Singh, A., Pra-

sad, R. and Sá-Correia, I., “The yeast ABC trans-

porter Pdr18 (ORF YNR070w) controls plasma

membrane sterol composition, playing a role in mul-

tidrug resistance”, Biochemical Journal, 440(2):

195-202, 2011.

[4] Cabrito, T.R., Remy, E., Teixeira, M.C., Duque,

P., Sá-Correia, I., “Resistance to herbicides in the

model organisms Saccharomyces cerevisiae and

Arabidopsis thaliana: the involvement of multidrug

resistance transporters” In: Herbicides and Envi-

ronment, (Kortekamp A, Ed.), INTECH, Vienna,

Austria, 623-640, 2011.

Tissue and subcelullar localization of a plant MFS protein and

effect of this protein in the resistance to the herbicide 2,4-D

(collaboration with PMB-IGC)

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Objectives

The activities of the BSRG in the area of Environ-

mental Biotechnology and Chemistry are devel-

oped mainly in the fields of Environmental Microbi-

ology and Environmental Toxicology, aiming:

1. To improve knowledge on microbial biodegra-

dation of s-triazine herbicides, mainly focused on

terbuthylazine which in the last years, in the EU,

has replaced the herbicidal substance atrazine

hence gaining agricultural and ecotoxicological

relevance. Ultimate goal is to optimize bioremedia-

tion strategies that will help to detoxify soils pollut-

ed with s-triazine-based herbicidal formulations

(due mainly to overuse, careless disposal or acci-

dental spills), thus preventing contamination of ad-

jacent freshwater compartments with the herbicides

and their toxic chlorinated metabolites.

2. To characterize the microbial diversity and the

population dynamics inside sludge bed anaerobic

reactors used for the treatment of wastewater from

dairy industry.

3. To identify potential molecular biomarkers of

toxicity and to gain insights into the mechanisms of

response to xenobiotic compounds, in particular

pesticides in the eukaryotic model Saccharomyces

cerevisiae, based on genome-wide approaches.

4. To develop yeast-based bioassays for the

assessment of the toxicity of xenobiotics (e.g. pesti-

cides, synthetic dyes and others).

Research Topics

Bioremediation strategies for soils contaminated

with herbicidal formulations containing s-triazines –

the catabolic ability of selected s-triazine–

degrading strains (namely, Pseudomonas sp. ADP

and Arthrobacter aurescens TC1) is being exploited

envisaging efficient biodegradation of s-triazine

herbicides in contaminated environments. Studies

of the influence of nutrient limitations (e.g. C, N

sources) and/or of relevant environmental factors

that may vary in the field (e.g. pH, moisture, soil

aging, presence of other xenobiotics that may be

mixed with s-triazines in polluted sites) on s-

triazines biodegradation in soil by Pseudomonas

sp. ADP and Arthrobacter aurescens TC1, either

alone or in consortium, are underway. Information

gathered with these studies is being used to opti-

mize bioremediation strategies (bioaugmentation

and/or biostimulation) for the cleanup of soils

spiked with s-triazines-based herbicidal formula-

tions at doses mimicking situations arising from

recommended agricultural application up to worst-

case scenarios of soil contamination, in soil micro-

cosms where the potential for natural attenuation is

low. The potential efficacy of cleanup treatments,

at laboratory and at semi-field scales, is examined

at the ecotoxicological level (collaboration with

IMAR/Univ. Coimbra).

Environmental Microbiology and Toxicology

Cristina A. Viegas, Jorge H. Leitão, Isabel Sá-Correia, Miguel C. Teixeira, Arsénio M. Fialho,

Sílvia A. Sousa, Paulo J. Dias

PhD, MSc students and Research assistants: Fátima Nunes Gil, Christian G. Ramos, André M. Grilo, Vera

Lúcia da Silva, Carla Alexandra Mateus, Tânia R. Cabrito

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Molecular characterization of microbial communities

in UASB reactors – The molecular characterization

of microbial populations from UASB reactors oper-

ated continuous or intermittently (collaboration with

CESAM and DEP - Univ. Aveiro), is being per-

formed based on a set of oligonucleotide primers

targeting the 16S rRNA gene sequences of the Ar-

chaeal phyla Euryarchaeota or Crenarchaeota, and

the Eubacterial phyla Firmicutes, Bacteroidetes,

Chloroflexi, Proteobacteria, Deferibacteres or Spiro-

chaetes. The microbial population dynamics associ-

ated with changes in the reactors´ operational pa-

rameters is being assessed based on FISH method-

ologies using oligonucleotides targeting specific

microbial subpopulations.

Transcriptional profiling in Saccharomyces cere-

visiae relevant for predicting mechanisms and iden-

tifying biomarkers of pesticide toxicity - Genome-

wide expression profiling in the yeast S. cerevisiae

in response to exposure to equitoxic concentrations

(20%-inhibition of growth) of six pesticides from

different chemical families (chloroacetanilide, phe-

nylurea, chlorophenoxyacetic acid, carbamate and

anylinopyrimidine families) is being used to identify

molecular biomarkers of exposure and toxicity

which may be useful for development of yeast-

based bioassays for preliminary toxicity testing or

for assessment of pesticide toxicity in environmental

samples. Comparison of the datasets of differential-

ly expressed genes have highlighted the potential of

the transcriptional profiling approach to distinguish

between the toxicological effects of structurally dif-

ferent pesticides in the yeast, and is being exploited

to predict novel clues on yeast responses to pesti-

cide moderate toxicity that may be relevant for non-

target eukaryotes. Studies are in course to examine

relationships between the patterns of altered ex-

pression of candidate biomarker genes and the

effects at higher levels of biological organization

(such as, phenotypic effects in the yeast) or con-

ventional indices of toxicity to be obtained using

ecologically relevant toxicity tests, using environ-

mental samples simulated with soil-water (run-off)

test chambers (collaboration with IMAR/Univ. Coim-

bra).

Yeast- and Caenorhabditis elegans-based toxicity

testing – the single-cell eukaryotic model S. cere-

visiae (endpoints: growth inhibition; modification of

the expression of selected gene biomarkers) and

the simple animal model C. elegans (endpoint: inhi-

bition of reproduction) are being exploited for the

assessment of the potential toxicity of xenobiotics,

namely of pesticides and of synthetic dyes.

Yeast toxicometabolomics — Yeast is being ex-

plored to gain mechanistic insights into the toxicity

of, and tolerance to, the widely used agricultural

fungicide mancozeb, linked to the development of

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Parkinson’s disease and cancer as the result of

environmental exposure. During 2011 a Metabolom-

ics approach was used (see page 22 highlight) and

results are under analysis

Elucidation of the role of MDR transporters in pesti-

cide toxicity and resistance in yeast – The role and

regulation of multidrug efflux pumps in yeast re-

sistance to xenobiotic compounds is being exam-

ined, focusing their effect in membrane composition

(see page 15). The extrapolation of the gathered

knowledge on the MDR phenomenon in yeast to

more complex eukaryotes is underway, in particular

focusing Arabidopsis thaliana resistance to the

herbicide 2,4-D (see page 15 highlight).

Main Achievements

Scalling up to semi-field conditions of previous

bench-scale microcosms experiments with a biore-

mediation tool (bioaugmentation with Pseudomonas

sp. ADP and biostimulation with citrate) in soils con-

taminated with an atrazine commercial formulation

(mimicking misuse applications or accidental spills)

proved to enable effective removal of the herbicide

and reduction in the potential environmental risks of

atrazine for both soil and aquatic compartments in

just 7 days. An integrated approach was followed to

assess the soil habitat function and the soil retention

function using ecotoxicty tests with standard soil

and aquatic organisms in a soil-water simulator for

pesticide applications (collaboration with IMAR/Univ.

Coimbra) [1].

Global transcriptional profiling analyses in the

yeast model in response to herbicide concentrations

exerting slight to moderate effects at higher levels of

biological organization (namely, growth inhibition),

allowed to identify molecular biomarkers of expo-

sure and toxicity and to predict novel mechanistic

insights over potential toxicological effects of the

herbicide alachlor, a plant protection product of eco-

toxicological concern. Of particular interest was the

observation of a consistent trend of transcript levels

variation of several candidate biormarkers of

alachlor toxicity, measured by quantitative RT-PCR,

for herbicide concentrations causing comparatively

minor phenotypic effects [2].

Two simple and unexpensive toxicity-biossays

using the microbial eukaryotic model S. cerevisiae

BY4741 and the animal model C. elegans Bristol N2

were used to get an overview of the toxicological

effects of twenty distinct azo dyes and three model

wastewaters that mimic real dye-containing efflu-

ents. The level of detoxification achieved as a result

of biotransformation treatments using a sequential

enzymatic process with azoreductase plus laccase

or using whole-cells of recombinant bacteria co-

expressing both enzymes, was determined

(collaboration with ITQB) [3].

A new methodology, combining PCR, cloning,

and sequencing of clones discriminated by RFLP,

was used to characterize the microbial population in

anaerobic sludge bed reactors, operated in continu-

ous or intermittent modes [4, 5].

The action and regulation of the drug efflux

pumps Flr1 and Pdr18 upon yeast challenge with

the fungicide mancozeb and the herbicide 2,4-D,

respectively, were examined [6, 7]. A book chapter

focusing on the involvement of MDR transporters

from model organisms S. cerevisiae and A. thaliana

in herbicide resistance was published [8].

Funded projects

Studies on the efficacy and scale-up of bioremedi-

ation strategies for soils contaminated with the herb-

icide terbuthylazine, PTDC/AAC-AMB/111317/2009,

PI: Cristina A. Viegas

Development of bioassays for herbicide toxicity

based on gene expression profiling using yeast

cells, PTDC/AMB/64230/2006, PI: Cristina A. Vie-

gas.

Enzymatic degradation and synthesis of azo and

anthraquinonic dyes, PTDC/BIO/72108/2006, PI at

IST: Cristina A. Viegas.

Optimization and control of intermittent UASB re-

actors and microbial population dynamics, PTDC/

AMB/65025/2006, PI at IST: Jorge H. Leitão.

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Resistance to pesticides and other chemical

stresses of agricultural interest: Role of plant

Major Facilitator Superfamily Transporters,

PTDC AGR-AAM/102967/2008, PI at IST: Isabel

Sá-Correia.

Selected Publications

[1] Chelinho, S., Moreira-Santos, M., Silva, C., Cos-

ta, C., Viana, P., Viegas, C.A., Fialho, A.M., Ribeiro,

R., Sousa, J.P. “Semi-field testing of a bioremediation

tool for atrazine contaminated soils: evaluating the

efficacy on soil and aquatic compartments”, Environ-

mental Toxicology and Chemistry (accepted,

Feb2012).

[2] Gil, F.N., Gonçalves, A.C., Jacinto, M.J, Becker,

J.D., Viegas, C.A., “Transcriptional profiling in Saccha-

romyces cerevisiae relevant for predicting alachlor

mechanisms of toxicity”, Environmental Toxicology

and Chemistry, 30: 2506-2518, 2011.

[3] Mendes, S., Farinha, A., Ramos, C.G., Leitão,

J.H., Viegas, C.A., Martins, L.O., “Synergistic action of

azoreductase and laccase leads to maximal decolouri-

zation and detoxification of model dye-containing

wastewaters”, Bioresource Technology, 102: 9852-

9859, 2011.

[4] Nadais, H., Barbosa, M., Capela, I., Arroja, L.,

Ramos, C.G., Grilo, A., Sousa, S.A., Leitão, J.H.,

“Enhancing wastewater degradation and biogas pro-

duction by intermittent operation of UASB reactors”,

Energy, 36: 2164-2168, 2011.

[5] Ramos, C.G., Grilo, A.M., da Costa, P.J.P., Nada-

is, H., Leitão, J.H. , "Extraction and Purification of DNA

from UASB Reactors Sludge Samples". In: DNA Bind-

ing and DNA Extraction: Methods, Applications and

Limitations (Chunxu Zhou and Xia Ling Eds.). Nova

Publishers. 2011. ISBN 978-1-61470-958-9.

[6] Monteiro, P.T., Dias, P.J., Ropers, D.,

Oliveira, A.L., Sá-Correia, I., Teixeira, M.C.,

Freitas, A.T., “Qualitative modelling and formal

verification of the FLR1 gene mancozeb response

in Saccharomyces cerevisiae”, IET Systems Biol-

ogy, 5(5): 308-16, 2011.

[7] Cabrito, T.R., Teixeira, M.C., Singh, A., Pra-

sad, R. and Sá-Correia, I., “The yeast ABC trans-

porter Pdr18 (ORF YNR070w) controls plasma

membrane sterol composition, playing a role in

multidrug resistance”, Biochemical Journal, 440

(2): 195-202, 2011.

[8] Cabrito, T.R., Remy, E., Teixeira, M.C., Duque,

P., Sá-Correia, I., “Resistance to herbicides in the

model organisms Saccharomyces cerevisiae and Ara-

bidopsis thaliana: the involvement of multidrug re-

sistance transporters”, In: Herbicides and Environ-

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Proposed model for the action of mancozeb in S. cerevisiae cells.

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Objectives

The emerging transdisciplinary field of Toxicoge-

nomics aims to study the cell response to a given

toxicant at the genome, transcriptome, proteome

and metabolome levels. The use of model organ-

isms to gather such genomic information, through

the exploitation of Omics and Bioinformatics ap-

proaches and tools, together with more focused

molecular and cellular biology studies are rapidly

increasing our understanding and providing an inte-

grative view on how cells interact with their environ-

ment. Despite the limitations intrinsic to the use of

such a simple single cell experimental model, the

yeast Saccharomyces cerevisiae is one of the most

interesting systems to obtain a truly global under-

standing of the toxicological response and re-

sistance mechanisms, being in the frontline of sys-

tems biology research and developments.

Our research in yeast toxicogenomics during 2011

was applied to study the genome-wide response to

chemical stresses that have an impact on:

1. Environmental health - predict toxicological

outcomes of exposure to environmental pollutants/

pesticides, find targets for genetic manipulation to

increase crop robustness, identify pesticide off-

targets and the relationship with human diseases.

2. Medicinal research and drug development -

elucidate mechanisms of drug action, identify direct

and off-target effects of dugs, and identify predic-

tive biomarkers of drug toxicity.

3. Biotechnology - identify predictive biomarkers

of industrial process outcome, and find targets for

genetic manipulation to increase microbial strain

robustness for biotechnological processes.

Research Topics

1. Yeast toxicogenomics applied to environ-

mental health

The toxicological outcome of sudden or chronicle

exposure to environmental pollutants and other

xenobiotic compounds (e.g. pesticides) used in

agriculture is scarcely understood at the molecular

level. Moreover, the exact toxicological outcome of

exposure to agrochemicals, such as herbicides and

agricultural fungicides, is difficult to predict, since

many times it takes years to develop.

Yeast toxicogenomics has been applied to define

and predict new toxicological outcomes of expo-

sure to mancozeb, an agricultural fungicide linked

to the development of Parkinson’s disease and

cancer as the result of environmental exposure,

Yeast Toxicogenomics:

Genome-wide responses to chemical stress

Isabel Sá-Correia, Miguel C. Teixeira, Nuno P. Mira, Cristina A. Viegas, Sandra C. dos Santos,

Margarida Palma, Paulo J. Dias

PhD and MSc students: Tânia R. Cabrito, Artur B. Lourenço, Filipa C. Roque, Sílvia F. Henriques, Joana F.

Guerreiro, Filipa Valada, Cláudia S. Godinho, Filipe Silva, Fátima Gil

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using expression proteomics (Santos et al., 2009,

Proteomics, 9: 657-70) and chemogenomics (Dias

et al., 2010, OMICS, 14: 211-27), and to the herbi-

cide 2,4-D, using expression proteomics (Teixeira

et al., 2005, Proteomics, 5: 1889-901) and tran-

scriptomics (Teixeira et al., 2006, FEMS Yeast

Res, 6: 230-48). The results obtained in yeast have

been used to guide more focused studies on the

molecular mechanisms underlying 2,4-D toxicity

and response in plants and other higher eukaryotes

(Cabrito et al., 2009, Appl Microbiol and Biotechnol,

84: 927-36, and unpublished results).

2. Pharmacogenomics studies

The use of yeast as a eukaryotic model is particu-

larly important in the field of medicinal research and

drug discovery. In this context, our group has ap-

plied yeast toxicogenomics approaches to the

study of the antimalarial quinine, namely chemoge-

nomics [1] and transcriptomics (dos Santos et al.,

2009, Antimicrob Agents Chemother, 53: 5213-23),

and to the study of the anticancer drug imatinib, in

specific chemogenomics (dos Santos and Sá-

Correia, 2009, Omics, 13: 185-98) and expression

proteomics (submitted results, see page 8).

A transcriptomic analysis had previously unveiled

an early glucose de-repression response to quinine

in yeast (dos Santos et al., 2009, Antimicrob

Agents Chemother, 53: 5213-23). Moreover, qui-

nine was shown to inhibit the uptake of glucose into

yeast cells following a competitive inhibition kinetic

model. These findings have an important parallel in

the malaria parasite, where glucose uptake is vital

and mediated by PfHT1, a single-copy transporter

homologous to yeast’s. These results were comple-

mented by the recent chemogenomics analysis [1].

Likewise, the yeast vacuolar H+-ATPase (V-

ATPase) had been identified in yeast cells as a

potential new target of imatinib (dos Santos and Sá

-Correia, 2009, Omics, 13: 185-98), and this study

was complemented by a quantitative- and phospho

-proteomics approach (submitted results).

3. Yeast toxicogenomics applied to fermenta-

tion- and industrial-related stresses

S. cerevisiae has been used for millennia in fer-

mentation processes and is also a preferable sys-

tem for the production of bioethanol. Furthermore,

S. cerevisiae can be used as a cell factory for the

production of a number of interesting biomolecules

with biotechnological and pharmaceutical interest.

In all these industrial processes, yeast cells have to

cope with stressing environmental conditions, in-

cluding chemical stress coming from the raw mate-

rial composition, and from the accumulation of eth-

anol, weak acids and other toxic by-products of the

yeast metabolism.

Toxicogenomics tools have been used with suc-

cess to characterise the toxicological outcome of

yeast exposure to fermentation-related chemical

stress inducers. Such an approach has the poten-

tial to elucidate the mechanisms of yeast tolerance

to fermentation stressors, thus providing clues on

how to improve process conditions and to engineer

yeast strains to increase fermentation yield. In addi-

tion, similar approaches have been applied to the

field of food production and preservation, to identify

molecular targets for new food preservation strate-

gies and find predictive biomarkers of food produc-

tion process outcome (in more detail in page 15).

Main Achievements

The knowledge based on the exploitation of

toxicogenomics in the yeast model were integrated

an invited review article where many of our toxi-

cogenomics-related results are discussed [1].

A chemogenomics-based analysis identified for

the first time several genes encoding ribosome

protein subunits whose deletion leads to increased

quinine resistance [4]. The particular involvement of

phosphate signalling and transport in quinine toler-

ance was also studied, with indications that phos-

phate-starvation responsive genes are activated in

response to quinine. Plasmodium falciparum ho-

mology searches identified several relevant func-

tional homologs, suggesting that quinine targets

identified in the yeast model are good candidates to

be transposed and explored in a parasitic context.

The response and resistance mechanisms to

herbicides in the model eukaryote S. cerevisiae

and the model plant Arabidopsis thaliana, in partic-

ular those involving MDR transporters (see page

18), including many of our recent findings in yeast,

were published as a book chapter [2].

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Transcriptional profiling analyses in the yeast

model in response to herbicide concentrations ex-

erting slight to moderate phenotypic effects

(namely, growth inhibition) was used to identify

molecular biomarkers of exposure and toxicity and

to predict novel mechanistic insights into the herbi-

cide alachlor toxicity, of ecotoxicological concern [3

and unpublished results] (see page 18).

A 2-DE based approach (see page 8) was

used to identify proteins altered at the content level

or displaying imatinib-repressed phosphorylation

(submitted results). The proteins identified have

human homologs and are mainly involved in glyco-

lytic pathways, translation and protein folding.

Imatinib was shown to act as a potent inhibi-

tor of the highly conserved yeast V-ATPase, both in

vivo and in vitro (dos Santos and Sá-Correia, 2009,

Omics, 13: 185-98, and unpublished results), sug-

gesting that vacuolar function is a novel imatinib

target.

Metabolic changes occurring in the presence

of the widely used bakery and fresh dairy products

preservative propionic acid, using high-resolution 1H-NMR spectroscopy were identified (see page 8)

[5].

Characterisation of the proteome-wide chang-

es occurring in the acetic acid highly-resistant yeast

species Z. bailii when exposed to growth inhibitory

but sub-lethal concentrations of acetic acid [6] (see

page 8).

Candidate genes for yeast engineering were

identified to improve improve bioethanol production

in Very-High-Gravity and lignocellulosic biomass

industrial fermentations [8].

Funded Projects

Resistance to pesticides and other chemical stress-

es of agricultural interest: Role of plant Major Facili-

tator Superfamily Transporters, PTDC/AGR-

AAM/102967/2008, PI at IST: Isabel Sá-Correia.

ZygoSacAR - Mechanistic insights into acetic acid

resistance in food spoilage yeasts: from the experi-

mental Saccharomyces cerevisiae to Zygosaccha-

romyces spp., PTDC/AGR-ALI/102608/2008, PI:

Isabel Sá-Correia.

INTACT - Integral Engineering of Acetic Acid Toler-

ance in Yeast, in the frame of the ERA-NET Indus-

trial Biotechnology, ERA-IB/0002/2010, PI: Isabel

Sá-Correia.

Imatinib resistance and targets in chronic myeloid

leukaemia: post-genomic approaches using yeast

as a model system, PTDC/SAU-FCF/71760/2006,

PI: Isabel Sá-Correia.

Development of bioassays for herbicide toxicity

based on gene expression profiling using yeast

cells, PTDC/AMB/64230/2006, PI: Cristina A. Vie-

gas.

Selected Publications

[1] dos Santos, S.C., Teixeira, M.C., Cabrito,

T.R., and Sá-Correia, I. “Yeast Toxicogenomics:

genome-wide responses to chemical stresses with

impact in Environmental Health, Pharmacology and

Biotechnology”, submitted to Frontiers in Genet-

ics upon invitation.

[2] Cabrito, T.R., Remy, E., Teixeira, M.C., Du-

que, P., and Sá-Correia, I., "Resistance to herbi-

cides in the model organisms Saccharomyces

cerevisiae and Arabidopsis thaliana: the involve-

ment of multidrug resistance transporters," in Herb-

icides and Environment, ed. A. Kortekamp.

(Vienna, Austria: INTECH), 623-640, 2011.

[3] Gil, F.N., Gonçalves, A.C., Jacinto, M.J,

Becker, J.D., and Viegas, C.A., “Transcriptional

profiling in Saccharomyces cerevisiae relevant for

predicting alachlor mechanisms of toxicity”, Envi-

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20 11 Annual Report

ronmental Toxicology and Chemistry, 30: 2506-

2518, 2011.

[4] dos Santos, S.C., and Sá-Correia, I.,”A ge-

nome-wide screen identifies yeast genes required

for protection against or enhanced cytotoxicity of

the antimalarial drug quinine”, Molecular Genetics

and Genomics 286: 333-346, 2011.

[5] Lourenço, A.B., Ascenso, J.R., and Sá-

Correia, I., “Metabolic insights into the yeast re-

sponse to propionic acid based on high resolution 1H-NMR spectroscopy”, Metabolomics 7: 457-468,

2011.

[6] Guerreiro, J.F., Mira, N.P., and Sá-Correia, I.,

“Adaptive response to acetic acid in the highly re-

sistant yeast species Zygosaccharomyces bailii

revealed by quantitative proteomics”, under revi-

sion in Proteomics.

[7] Teixeira, M.C., Mira, N.P., and Sá-Correia, I.,

“A genome-wide perspective on the response and

tolerance to food-relevant stresses in Saccharomy-

ces cerevisiae”, Current Opinion in Biotechnolo-

gy 22: 150-156, 2011.

[8] Pereira, F.B., Guimarães, P.M., Gomes, D.G.,

Mira, N.P., Teixeira, M.C., Sá-Correia, I., and

Domingues, L., “Identification of candidate genes

for yeast engineering to improve bioethanol produc-

tion in Very-High-Gravity and lignocellulosic bio-

mass industrial fermentations”, Biotechnology for

Biofuels 4: 57, 2011.

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ment (Kortekamp A, Ed), INTECH, Vienna, Austria,

p.623-640, 2010 (ISBN:978-953-307-476-4).

Objectives

Extracellular polysaccharides (EPS) of bacterial origin

have diverse functions in nature, such as in bacterium

-plant interactions, virulence factors or protective

agents. Due to their unique properties as rheology

modification agents, stabilizers, emulsifiers and gel-

ling agents, they represent an important class of poly-

meric materials with interest in Biotechnology. Re-

search carried out by our group is focused on the

proteins directing EPS biosynthesis, on EPS-

mediated interaction with hosts, either in symbiosis or

pathogenesis, and on EPS potential industrial appli-

cations. The biological systems under study are the

exopolysaccharide cepacian from Burkholderia and

the exopolysaccharides succinoglycan and galac-

toglucan from Sinorhizobium meliloti.

Research topics

1. Mucoid morphotype variation of Burkholderia multi-

vorans during chronic persistence in the airways of

cystic fibrosis patients

Long-term infection of the airways of Cystic Fibrosis

(CF) patients with Burkholderia cepacia complex bac-

teria has been associated with emergence of pheno-

type variation. We studied two Burkholderia multi-

vorans clonal isolates displaying different mor-

photypes from a chronically infected CF patient to

evaluate traits development during lung infection.

Expression profiling of mucoid D2095 and nonmucoid

D2214 isolates using custom Burkholderia microar-

rays revealed downregulation of genes encoding

products related to virulence-associated traits and

metabolism in D2214. Furthermore, D2214 showed

no exopolysaccharide production, lower motility and

chemotaxis, lower virulence in Galleria mellonella,

and more biofilm formation. Mucoids vs. nonmucoid

morphotype triggering cues identified include high

antibiotic concentration, osmotic and oxidative stress-

es and nutrient limitation. Overall, adaptation during

Bcc chronic lung infections give rise to genotypic and

phenotypic variation among isolates, contributing to

their fitness while maintaining their capacity for surviv-

al in this opportunistic human niche.

2. Proteins required for exopolysaccharide biosynthe-

sis in Burkholderia cepacia complex

BceF protein from B. cepacia IST408 belongs to the

bacterial tyrosine kinase family of proteins known to

regulate polysaccharide biosynthesis, DNA metabo-

lism and stress responses. Despite its known involve-

ment in the biosynthesis of the exopolysaccharide

cepacian, we had shown that a bceF mutant is im-

paired in stress response, swimming motility, biofilm

formation and virulence. In addition, the structure of

BceC, a UDP-glucose dehydrogenase (UGD) enzyme

Bacterial exopolysaccharides:

biosynthesis and biological role

Leonilde M. Moreira, Arsénio M. Fialho, Jorge H. Leitão, Sílvia A. Sousa, Ana S. Ferreira,

Isabel Sá-Correia

PhD and MSc students: Inês N. Silva, Mário A. Santos; Andreia T. Marques, Joana R. Feliciano, Vitor H.

Oliveira, Andreia C. Tavares, Sílvia Matos, Filipa G. Dias, Daniela Isidoro

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20 11 Annual Report

responsible for the NAD-dependent 2-fold oxidation

of UDP glucose to UDP-glucuronic acid, was deter-

mined at 1.75-Å resolution. Mutagenic studies were

performed on the BceC glycine-rich motif

(GXGYXG) of its N-terminal Rossmann-like domain

and Y10 was shown to be a key catalytic residue in

the final hydrolysis of the enzymatic thioester inter-

mediate. The BceN protein with GDP-D-mannose

dehydratase activity, involved in the first step of the

enzymatic conversion of GDP-D-mannose into GDP

-D-rhamnose, is being functionally characterized.

3. Sinorhizobium meliloti proteins with a role in ex-

opolysaccharide biosynthesis and in nitrogen fixa-

tion symbiosis

Members of the TetR family of repressors are

known to control genes whose products are in-

volved in multidrug resistance, catabolic pathways,

biosynthesis of antibiotics, osmotic stress and path-

ogenicity. We have characterized a TetR member of

S. meliloti encoded by gene SMc03169. A deletion

mutant exhibited lower tolerance to acidic condi-

tions and increased susceptibility to detergents and

oxidative stress agents. This mutant also showed

defects in symbiosis with Medicago sativa, since the

number of pink nodules decreased by one-half and

it was unable to directly compete with the wild-type

strain for nodulation. Expression profiling showed

downregulation of many genes involved in Nod fac-

tor and siderophore biosynthesis, phosphate uptake

systems, and genes belonging to the heat-shock

regulon. Among the upregulated genes in the mu-

tant we found genes directing succinoglycan bio-

synthesis, lipopolysaccharide modification and

genes whose products are involved in metabolism.

Main achievements

Evidences on the genotypic and phenotypic

traits variation of Burkholderia cepacia complex

bacteria during chronic lung colonization.

Demonstration that the BceF tyrosine kinase

from Burkholderia cepacia is required for exopoly-

saccharide biosynthesis, stress response, cell motil-

ity, biofilm formation and virulence.

Determination of the crystal structure of UDP-

glucose dehydrogenase BceC from B. cepacia in

collaboration with the Protein Crystallography group

of ITQB.

Demonstration that the Sinorhizobium meliloti

TetR repressor encoded by SMc03169 is involved

in regulatory networks targeting cell envelope and

metabolism and is required for the biological nitro-

gen fixation symbiosis with Medicago sativa.

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Funded projects

KINASE- Phosphoproteomics in Burkholderia

to assess the role of tyrosine phosphorylation in

virulence determinants expression, PTDC/QUI-

BIQ/118260/2010, PI: Leonilde M. Moreira.

ROOT-INT– Role of a two-component regula-

tory system in the early interaction between Sino-

rhizobium meliloti and plant root hairs, PTDC/BIA-

MIC/113733/2009, PI: Leonilde M. Moreira.

Exploiting the type II phosphomannose isomer-

ise BceAJ as a new target for the development of

new antimicrobials and for biotechnological applica-

tions, PTDC/EBB-BIO/098352/2008, PI: Jorge H.

Leitão.

Selected publications

[1] Silva IN, Ferreira AS, Becker JD, Zlosnik

JEA, Speert DP, He J, Mil-Homens D, and

Moreira LM., “Mucoid morphotype variation of

Burkholderia multivorans during chronic cystic fi-

brosis lung infection is correlated with changes in

metabolism, motility, biofilm formation and viru-

lence”, Microbiology, 157:3124-3137, 2011.

[2] Ferreira AS, Silva IN, Oliveira VH, Cunha

R, and Moreira LM., “Insights into the role of extra-

cellular polysaccharides in Burkholderia adaptation

to different environments”, Frontiers in Cellular

and Infection Microbiology. 1:16, 1-9, 2011 doi:

10.3389/fcimb.2011.00016.

[3] Rocha J, Popescu AO, Borges P, Mil-

Homens D, Moreira LM, Sá-Correia I, Fialho AM,

Frazão C., “Burkholderia cepacia UDP-glucose

dehydrogenase (BceC) structure and the role of

Tyr10 in the final hydrolysis of UGDs thioester in-

termediate”. Journal of Bacteriology, 193:3978-

3987, 2011

[4] Ferreira, A.S., Silva, I.N., Moreira, L.M.,

“Mechanisms controlling the expression of the ex-

opolysaccharide of Burkholderia and role in niche

adaptation”, In: Biopolymers (M Elnashar, Ed)

INTECH, Vienna, Austria (ISBN 978-953-307-179-

4), 2011.

[5] Sousa, S.A., Feliciano, J., Leitão, J.H.,

“Sugar nucleotides: biosynthetic pathways and

biological roles of an important class of intermedi-

ate metabolites in bacteria”. In: Biopolymers (M

Elnashar, Ed) INTECH, Vienna, Austria (ISBN 978-

953-307-179-4), 2011.

[6] Ferreira AS, Silva IN, Becker JD, and

Moreira, LM., “Role of tyrosine phosphorylation in

the regulation of Burkholderia cell physiology”, Bio-

engineering (ENBENG), 2011. 1st Portuguese Meet-

ing, vol. no. 1-4 March pp.1-4. doi:10.1109/

ENBENG.2011.6026056.

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20 11 Annual Report

Objectives

Long-term respiratory infections with Burkholderia

cepacia complex (Bcc) bacteria in cystic fibrosis

(CF) patients generally lead to a more rapid decline

in lung function and, in some cases, to a fatal ne-

crotizing pneumonia known as the “cepacia syn-

drome”. Bcc bacteria are serious opportunistic path-

ogens that are virtually impossible to eradicate from

the CF lung, posing a serious clinical threat. During

chronic infection, the CF airways represent an

evolving ecosystem, with multiple phenotypic vari-

ants emerging from the clonal population and be-

coming established in the patients’ airways as the

result of genetic adaptation.

In this context, the global objective of our research

during 2011 was the study and characterisation of

mechanisms of persistence and adaptation of Bcc

bacteria in the lung of CF patients, through different

approaches - phenotypic characterisation, ge-

nomics, metabolomics and comparative genome-

wide expression analyses (transcriptomics and ex-

pression proteomics) of different Bcc clinical iso-

lates.

Research topics

1. Epidemiology of Bcc bacteria in a Portuguese CF

subpopulation

The epidemiological survey of Bcc bacteria involved

in respiratory infections at the major Portuguese CF

Treatment Centre at Santa Maria Hospital, in Lis-

bon, has been carried out by our research group for

the past 16 years, covering over 500 clinical isolates

where B. cepacia and B. cenocepacia are the pre-

dominant species and B. stabilis, B. contaminans,

B. dolosa and B. multivorans are also represented.

The systematic and longitudinal survey of this CF

population during such an extended period of time

represents a unique case study, comprehending 41

Bcc-infected patients (29 paediatric and 12 adult) of

whom around 70% have been persistently colonised

between 7 months and 9 years (Richau et al., 2000,

J Clin Microbiol, 38:1651-5; Cunha et al., 2003, J

Clin Microbiol, 41:4113-20; Cunha et al., 2007, J

Clin Microbiol, 45:1628-33).

Although B. cepacia is not a predominant Bcc spe-

cies among the CF population characterised world-

wide, our analyses unveiled an exceptionally high

incidence of this species at the HSM CF Centre

from 2003 to 2005 (Cunha et al., 2007, J Clin Micro-

biol, 45:1628-33). This abnormal prevalence of B.

cepacia was associated with a contamination de-

tected by the Portuguese Medicines and Health

Products Authority (INFARMED) in non-sterile sa-

line solutions for nasal application. Finally, although

most of our epidemiologic studies have focused on

the 2 most prevalent species, we have recently doc-

umented the first case of B. dolosa chronic infection

in this CF centre, with a period of transient co-

infection with B. cenocepacia.

2. Evolution within the CF lung

During chronic colonisation of the CF airways, mi-

crobial pathogens undergo widespread positive se-

lection across the genome in response to stressing

pressures exerted by the host environment, in par-

ticular those resulting from challenges of the im-

Burkholderia cepacia complex bacteria in cystic fibrosis: epidemiology, clonal variation and

genome-wide expression alterations

Isabel Sá-Correia, Carla C. Coutinho, Sandra C. dos Santos, Nuno P. Mira

PhD and MSc students: Andreia Madeira, Ana S. Moreira, Rita Maldonado

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mune defences, antimicrobial therapy, nutrient avail-

ability and oxygen limitation. Although recent stud-

ies have tried to elucidate the topic of microbial evo-

lution within the CF lung, the focus has been mainly

on Pseudomonas aeruginosa, while equivalent stud-

ies on Bcc bacteria remain lacking. A better under-

standing of how Bcc bacteria adapt to the stressing

CF lung environment is crucial to deal with these

chronic infections.

Our research activity has focused on the study of

these adaptive mechanisms based on extensive

phenotypic, genotypic and genome-wide expression

approaches of selected Bcc clonal variants obtained

during long-term colonisation of the CF airways.

These studies have been primarily dedicated to a

series of 11 clonal isolates retrieved from the same

patient from the onset of Bcc infection to his death

3.5 years later with the cepacia syndrome, in partic-

ular IST439, the isolate believed to have initiated

the infection, IST4113, recovered almost 3 years

later after a period of exacerbated infection and

intravenous therapy, and IST4134, retrieved from

the patient immediately before his death [1-4].

3. Adaptation to microaerophilic metabolism

Bacteria are able to grow to high densities in the CF

lung in a material that is limited or depleted in oxy-

gen. Very low oxygen availability is currently recog-

nised as an important factor contributing to antibiotic

resistance and persistent infections of P. aeruginosa

in the CF airways, where the predominant mode of

growth is believed to be microaerophilic respiration,

but little is known about the underlying molecular

mechanisms.

Previous results obtained by our group [ref. 3 and

unpublished results] suggest that mechanisms in-

volved in chronic infection (e.g. genetic expression,

antibiotic resistance, biofilm formation, membrane

fatty acid composition, etc.) are altered in isolates

retrieved during a stage of advanced lung deteriora-

tion, when presumably the amount of oxygen availa-

ble is very low. Metabolomics (exo- and intra-

cellular) and expression proteomics (intracellular,

membrane and secretome) analyses of Bcc isolates

grown under low oxygen conditions are currently

underway (see page 8). In addition, we are currently

screening a B. cenocepacia J2315 mini-Tn5 Tel

(TelR) mutant collection to identify genes required

for microaerophilic growth.

Main achievements

A phenotypic assessment of 11 clonal variants

obtained from the same CF patient sputum during

3.5 years was carried out, including assays of: sus-

ceptibility against different classes of antimicrobials,

cell motility, cell hydrophobicity and zeta potential,

colony and cell morphology, fatty acid composition,

growth under iron limitation/load conditions, exopol-

ysaccharide production and size of the biofilms

formed. The results are suggestive of clonal expan-

sion of B. cenocepacia during long-term colonisation

[3].

Description of an adaptive strategy to severely

oxygen-limited conditions involving a reduction of

the fatty acid saturation degree that occurs with

deterioration of pulmonary function. The degree of

fatty acid saturation observed in isolates grown un-

der microaerophilic conditions is significantly below

the one exhibited by isolates grown under aerophilic

conditions [3].

Identification of 79 proteins that are differen-

tially expressed between two sequential B. cenoce-

pacia isolates, IST439 and IST4113. These proteins

are mainly involved in carbohydrate metabolism,

translation, iron uptake, nucleotide synthesis, pro-

tein folding, peptidoglycan, membrane lipids and

lipopolysaccharide synthesis [1].

A similar approach was adopted for the com-

parison of IST439 and IST4134, with the identifica-

tion of 53 proteins (unpublished results). The quanti-

tative comparisons of the different proteomes sug-

gests a genetic adaptation leading to bacterial per-

sistence in the CF airways.

Identification of over 1000 differentially ex-

pressed genes in IST439 and IST4113. The results

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20 11 Annual Report

are similar, although more comprehensive, to the

ones obtained with the quantitative proteomics ap-

proach. Alterations related with adaptation to the

nutritional environment of the CF lung and to an

oxygen-limited environment are also suggested to

be a key feature of transcriptional reprogramming

occurring during long-term colonisation, antibiotic

therapy and disease progression [4].

Full genome sequencing of four sequential

clonal isolates retrieved from the same CF patient is

being concluded. The integrated analysis of the

resulting sequences is expected to provide clues

into the genomic evolution underwent by these iso-

lates during chronic colonisation of the patient’s

lung.

Funded Projects

ADHRES - Signature Project: Expression profiling of

adhesive (AHD) and resistance (RES) genes in

biofilm lifestyle in P. aeruginosa, P. putida and B.

cenocepacia, in the frame of the ERA-NET Patho-

genomics, ERA-PTG/SAU/0001/2008, PI: Isabel Sá

-Correia.

COST BM1003 action: Microbial cell surface deter-

minants of virulence as targets for new therapeutics

in Cystic Fibrosis, PI at IST: Isabel Sá-Correia.

Selected publications

[1] Madeira, A., Santos, P.M., Coutinho, C.P.,

Pinto-De-Oliveira, A., and Sa-Correia, I.,

“Quantitative proteomics (2-D DIGE) reveals molec-

ular strategies employed by Burkholderia cenocepa-

cia to adapt to the airways of cystic fibrosis patients

under antimicrobial therapy”, Proteomics 11: 1313-

1328, 2011.

[2] Coutinho, C.P., dos Santos, S.C., Madeira, A.,

Mira, N.P., Moreira, A.S., and Sa-Correia, I., “Long

-term colonization of the cystic fibrosis lung by

Burkholderia cepacia complex bacteria: epidemiolo-

gy, clonal variation, and genome-wide expression

alterations”, Frontiers in Cellular Infection and

Microbiology 1, 1-11, 2011.

[3] Coutinho, C.P., de Carvalho, C.C., Madeira,

A., Pinto-De-Oliveira, A., and Sá-Correia, I.,

“Burkholderia cenocepacia phenotypic clonal varia-

tion during a 3.5-year colonization in the lungs of a

cystic fibrosis patient”, Infection and Immunity 79,

2950-2960, 2011.

[4] Mira, N.P., Madeira, A., Moreira, A.S.,

Coutinho, C.P., and Sá-Correia, I., ”Genomic ex-

pression analysis reveals strategies of Burkholderia

cenocepacia to adapt to cystic fibrosis patients' air-

ways and antimicrobial therapy”, PLoS One 6,

e28831, 2011.

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Bacterial protein azurin as a new candidate drug to treat P-cadherin overexpressing breast cancer B

SR

G

This project is being pursued in collaboration with Dr

Joana Paredes and Dr Raquel Seruca, IPATIMUP,

Portugal, and aims to explore the use of a bacterial

protein named azurin, as a novel anti-cancer agent

[1,2]. Azurin is a low molecular weight (14kDa), wa-

ter-solube protein produced by the bacterium Pseu-

domonas aeruginosa. Besides its well documented

function as a redox partner in electron transfer reac-

tions, azurin has been found to have cytotoxicity

activity towards human cancer cell lines in vitro and

in vivo. It has been demonstrated that azurin can

enter preferentially into cancer cells and forms a

complex with the tumor suppressor p53, stabilizing it

and inducing apoptosis [Yamada et al. Cell Microbi-

ol, 7: 1418-1431, 2005; Fialho et al., Curr Opin Bio-

technol 2007, 18:279-286].

Azurin is an immunoglobulin (Ig)-like domain (single chain

format) and adopt a -fold with loops at the two poles that

allow a large binding interface.

One of the most important properties of azurin is its

ability to mediate high-affinity interactions with unre-

lated proteins, conferring on it the property of a nat-

ural scaffold for therapeutic purposes. In this pro-

ject, we aim at studying the interaction between

azurin and a specific type of Cadherin, the P-

cadherin. P-cadherin overexpression occurs in

about 30% of all breast carcinomas and has been

shown to promote invasion, motility and migration of

breast cancer cells, which in part is due to a soluble

form of this protein (sP-cadherin) [Ribeiro et al.,

Oncogene 2010, 29(3):392-402]. We hypothesized

that azurin could be a scaffold against P-cadherin,

antagonizing its pro-invasive effects.

P-Cadherin (green color) over-expressing breast cancer

cells: poor prognosis predictor. This protein is frequently

overexpressed in invasive breast carcinomas. Azurin could

be a scaffold against this protein, being an interesting new

therapeutic tool.

Main achievements:

Azurin causes a specific decrease of P-

cadherin in human breast cancer cell lines

In vitro, one single dose of azurin at 50 μM caused

a specific decrease in P-cadherin protein levels from

30-50% in two different cell lines. On the other

hand, the levels of E-cadherin, a known tumor sup-

pressor, remain unaltered or even increased. Addi-

tionally, the soluble form of P-cadherin (sP-cad) was

reduced after azurin treatment.

Azurin decreases P-cadherin expression in breast cancer

cells (Sum 149PT).Immunofluorescence analysis: the

green color, highlight the presence of P-cadherin in the

plasma membranes while the blue color represents the

nucleus.

Bacterial protein azurin as a new candidate drug to

treat P-cadherin overexpressing breast cancer BSR

G

Arsénio M. Fialho PhD student: Nuno Bernardes

33

20 11 Annual Report

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Azurin decreases invasion of MCF-7/Az.Pcad

and SUM 149 breast cancer cells

Matrigel invasion assays demonstrated that azurin

reduces the invasive phenotype of the cells, con-

cordant with the decrease on P-cadherin. We have

also observed a decrease in the matrix metallopro-

tease MMP2 activity in the extracellular media of

azurin treated cells.

Signaling pathways and matrix remodeling

triggered by azurin

Azurin led to a decrease in the phosphorylation lev-

els of both FAK and Src proteins, non-receptor tyro-

sine kinases activated by P-cadherin overexpres-

sion, involved in several signaling pathways that

regulate cell-cell and cell-matrix adhesion.

Altogether, our data show that azurin exhibits a spe-

cific preference for P-cadherin, abrogating its inva-

sive effects and, therefore, may have a potential

use in the treatment of breast carcinomas. We are

currently extending our studies aiming to gain a

better understanding about the molecular mecha-

nisms by which azurin interferes with P-cadherin in

breast cancer cells. We plan to perform gene ex-

pression microarray analysis of azurin treated and

untreated P-cadherin overexpressing cancer cells.

Moreover, we plan to perform further animal studies

to assess the potential use of azurin as a novel ther-

apeutic strategy to treat P-cadherin overexpressing

breast cancer.

Funded project:

Bacterial protein azurin as a new candidate drug to

treat poor-prognosis breast cancer, PTDC/EBB-

BIO/100326/2008, PI: Arsénio M. Fialho

Selected publications:

[1] Fialho A.M., Bernardes N., Chakrabarty A.M.

”Recent patents on live bacteria and their products

as potential anticancer agents” Recent Patents on

Anti-Cancer Drug Discovery 7: 31-55, 2012

[2] Fialho A.M., Salunkhe P., Manna S.K., Mahali

S., Chakrabarty A.M. “Glioblastoma Multiforme:

Novel Therapeutic Approaches” ISRN Neurology,

Article ID 642345, 2012

Issued patents

Fialho A.M., Bernardes N., Gonçalves J., Santos

A. C. “Compositions to treat HIV infections and

methods therefor” 1126/MUM/2011, March 2012

34

Small non-coding regulatory RNAs

and RNA chaperones in

Burkholderia cepacia complex virulence

Jorge H. Leitão and Sílvia A. Sousa

PhD and MSc students: Christian G. Ramos, André M. Grilo, Joana R. Feliciano, Paulo J.P. da Costa

BSR

G

Objectives

During the infection process, bacterial pathogens are

exposed to challenging conditions, promoted by the host

and the specific ecological niche. The ability to rapidly

adapt and to thrive under these stressing conditions

while expressing several virulence determinants, is de-

terminant for a successful infection. Burkholderia cepa-

cia complex (Bcc) bacteria are opportunistic multidrug

resistant human pathogens, able to cause life-

threatening respiratory infections mainly among cystic

fibrosis and immunocompromised patients. More recent-

ly these bacteria have emerged as important pathogens

among hospitalized patients suffering from several ma-

lignancies and morbidities, particularly among patients

suffering from diabetes and cancer. These bacteria pre-

sent unusually large genomes (~7000 genes), as well as

a surprisingly high number of predicted and expressed

sRNAs (Leitão et al., Appl Microbiol Biotechnol 2010,

87:31-40; and unpublished results).

The virulence of Burkholderia cepacia complex is multi-

factorial, requiring a wide array of distinct factors and

effectors (Leitão et al., Appl Microbiol Biotechnol 2010,

87:31-40). In other pathogens, virulence has been in-

creasingly shown to be regulated at the post-

transcriptional level by small non-coding regulatory

RNAs (sRNAs), often mediated by Hfq-like RNA chaper-

ones.

Work carried out by our group envisages the identifica-

tion and functional characterization of sRNAs and their

mRNA targets, particularly of those related to virulence

and resistance to stresses mimicking those faced by the

bacterium during the infection process. The research

work performed involves the isolation of sRNAS, the

construction of sRNA clone libraries, and the functional

characterization of those sRNAs related to virulence.

Our goal is to identify sRNAs and pathways which might

be exploited as targets for therapeutic intervention

against infections caused by these bacteria.

Research Topics

The research performed has been focused on the mo-

lecular identification and characterization of determi-

nants of virulence from Burkholderia cepacia complex

bacteria, and on the elucidation of the underlying molec-

ular mechanisms. An experimental strategy based on

the construction of mutant libraries from particularly viru-

lent B. cepacia complex strains, screening of attenuated

mutants using the nematode Caenorhabditis elegans as

an infection model, followed by cloning and functional

analysis of the genes identified has been pursued. Sev-

eral virulence-related genes were already described by

us, including the two Hfq-like RNA chaperones Hfq and

Hfq2. These two RNA chaperones are currently being

used in co-precipitation experiments to unveil small non-

coding regulatory RNAs that exert their regulatory effect

at the post-transcriptional level, mediated by RNA chap-

erones.

During 2011 our research was focused on:

1. RNOmics

sRNAs and Hfq-like RNA chaperones have been shown

to play important roles in bacterial virulence and re-

sistance to stress. sRNAs most often require Hfq-like

35

20 11 Annual Report

RNA chaperones both for their stability and for their

action. Hfq-like proteins bind tightly to the A/U rich

regions of RNAs, allowing for a selective enrich-

ment in these molecules, which then can be identi-

fied either by RNAseq or by cloning and sequenc-

ing. To date, 4 sRNAs have been validated alt-

hough not functionally characterized, despite the

more than 200 sRNAs bioinformatically predicted by

others to be encoded within the genome of

Burkholderia cenocepacia J2315. Ongoing research

envisages the experimental identification of Hfq- or

Hfq2-binding sRNAs expressed either under specif-

ic stress conditions or during infection of the nema-

tode Caenorhabditis elegans. Several sRNAs were

already identified by this strategy, some previously

predicted by bioinformatics, and novel ones that

escaped bioinformatics predictions.

2. Post-transcriptional control of expression of

virulence-related genes by sRNAs

Bacterial sRNAs can exert their regulatory activity

negatively by promoting their mRNA target rapid

decay in a ribonuclease-dependent fashion or by

blocking ribosome access to the mRNA target, or

positively by promoting the stability of the target

mRNA or by relieving inhibitory structures in the

mRNA. Hfq-like RNA chaperones are required for

the stability of sRNAs and their pairing with the re-

spective mRNA targets, with the exception of cis-

acting sRNAs, which pair with their mRNA targets

without protein intervention. Current research is

focused on the understanding of the roles of specif-

ic sRNAs in regulatory networks involved in the post

-transcriptional regulation of virulence-related

genes.

3. sRNA biology

The expression of sRNAs is generally highly transi-

ent, and occurs in response to very well defined

stimuli. The knowledge of the molecular mecha-

nisms involved in their biogenesis, processing, fold-

ing and stability, are of critical importance for the

understanding of their function, particularly of those

targeting multiple mRNAs. Current research is fo-

cusing on the identification of the molecular mecha-

nisms of sRNA expression, processing and stability,

and also on the molecular mechanisms of their ac-

tion.

4. RNA chaperones molecular biology

Bacteria of the Burkholderia cepacia complex are

among the few prokaryotes that have 2 functional

and distinct Hfq-like RNA chaperones. Both pro-

teins share a highly conserved core typical of Hfq-

like proteins, but differ in their C-terminus. Re-

search work performed so far suggest that despite

sharing some similar functions, each RNA chaper-

one might be involved in specific interactions, play-

ing distinct roles in the biology of Burkholderia ce-

pacia complex bacteria. We are currently focused

on the understanding of the roles played by the

distinct C- termini of Hfq and Hfq2 on the interac-

tions with both sRNAs and their mRNA targets, and

their consequences to the global expression of viru-

lence-related genes.

Main Achievements

The RNA chaperone Hfq2 from Burkholderia

cenocepacia J2315 was cloned and functionally

characterized. The results obtained indicate that

Hfq2 and the previously described Hfq play distinct

roles in cell physiology and virulence, in addition to

shared functions. This was the first study of a bacte-

rium harbouring two distinct and functional Hfq-like

proteins.

During 2011, we have developed an experi-

mental strategy to identify sRNAs from bacteria of

the Burkholderia cepacia complex, based on co-

precipitation of sRNAs with His-tagged Hfq and

Hfq2, followed by cloning and sequence analysis. A

total of 77 distinct sRNAs were already identified.

Comparative genomics showed that 11 of them are

unique to the Bcc.

The sRNAs h2cR and mtvR were functionally

characterized. h2cR is a cis-encoded negative regu-

lator of the mRNA levels of hfq2, depending on Hfq

for its stability. mtvR is a trans-encoded sRNA,

demonstrated to require Hfq2 for the negative regu-

lation exerted on hfq mRNA and on the mRNA lev-

els of 16 other genes. Manuscripts are presently

submitted for publication.

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36

The molecular characterization of the mtvR

sRNA was also performed. This work revealed the

details of the mtvR biogenesis and stability, as well

as the nucleotide sequence involved in base-pairing

with one of its targets.

Funded Projects

RNomics-Bcc: RNomics studies to identify small

non-coding RNAs of Burkholderia cepacia complex

involved in host-pathogen interactions, PTDC/BIA-

MIC/119091/2010, PI: Jorge H. Leitão.

Selected Publications

[1] Ramos CG, Sousa SA, Grilo AM, Feliciano

JR, Leitão JH, (2011) The second RNA chaperone

Hfq2, is also required for survival to stress and the

full virulence of Burkholderia cenocepacia J2315. J

Bacteriol, 193 (7):1515-152

[2] Ramos CG, Sousa SA, Grilo AM, da Costa

PJP, Feliciano JR, Leitão JH. (2011) A RNomics-

based strategy identifies regulatory small RNAs in

Burkholderia cepacia complex. Proceedings of the

First Portuguese Meeting in Bioengineering, 1-4

March, IST, TagusPark, Oeiras, Portugal.

[3] Sousa SA, Ramos CG, Leitão JH (2011)

Burkholderia cepacia Complex: Emerging Multihost

Pathogens Equipped with a Wide Range of Viru-

lence Factors and Determinants. Int J Microbiol.

pii: 607575.

[4]Ramos CG, Leitão JH. Caenorhabditis elegans

as a research tool to unveil bacterial virulence deter-

minants: Lessons from the Burkholderia cepacia

complex”. In: Nematodes: Morphology, Functions

and Management Strategies (Eds: F Boeri and JA

Chung). Nova Science Publishers. (ISBN 978-1-

61470-784-4). In press.

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-1400

-1200

-1000

-800

-600

-400

-200

0

200

400

600

190 200 210 220 230 240 250 260

Wavelength (nm)

[Θ]

(deg

cm

2d

mo

l-1)

-1400

-1200

-1000

-800

-600

-400

-200

0

200

400

600

190 200 210 220 230 240 250 260

Wavelength (nm)

[Θ]

(deg

cm

2d

mo

l-1)

Ald

olas

eBSA

Ova

lbum

ine

Hfq

2

kDa

158132

66

44

trimeric

monomeric

Ald

olas

eBSA

Ova

lbum

ine

Hfq

2

kDa

158132

66

44

trimeric

Ald

olas

eBSA

Ova

lbum

ine

Hfq

2

kDa

158132

66

44

trimeric

monomeric

Top Side

Conserved

RNA

binding

residues

Conserved

RNA

binding

residues

+ - + + ++ + + ++ Hfq

- + + ++ + + ++ + Hfq2

- - - - - + + + RNA

132 kDa

66 kDa

BSA

+ - + + ++ + + ++ Hfq

- + + ++ + + ++ + Hfq2

- - - - - + + + RNA

132 kDa

66 kDa

BSA

Kd ( M) 0.62 0.44 0.40 1.04 0.97 0.68

0.50 0.89

+ - + + ++ + + ++ Hfq

- + + ++ + + ++ + Hfq2

- - - - - + + + RNA

132 kDa

66 kDa

BSA

+ - + + ++ + + ++ Hfq

- + + ++ + + ++ + Hfq2

- - - - - + + + RNA

132 kDa

66 kDa

BSA

Kd ( M) 0.62 0.44 0.40 1.04 0.97 0.68

0.50 0.89

-helix

-sheet

Unstructured

region

37

20 11 Annual Report

Functional characterization of virulence-associated Trimeric Autotransporter Adhesins from the cystic

fibrosis pathogen Burkholderia cenocepacia

Arsénio M. Fialho PhD and MSc students: Dalila Mil-Homens, Eunice Penas

BSR

G

The Burkholderia cepacia complex (Bcc) consists of 17

related species that have been isolated from sputum of

Cystic Fibrosis (CF). The virulence of each species is

variable and B. cenocepacia, B. multivorans and B. dolo-

sa are the most problematic of these CF pathogens.

They have the capacity to disseminate and persist

among CF-patients and are resistant to many antimicro-

bial agents [1]. In a subset of CF patients, Bcc infections

lead to declining lung function, with necrotizing pneumo-

nia and a fatal septicemia termed “cepacia syn-

drome” [Mahenthiralingam et al., Nat Rev Micro 3:144-

156, 2005].

The genome sequence of the epidemic B. cenocepacia

strain J2315, an ET-12 member, has been determined

[Holden et al., J Bact 191: 261-277, 2009]. Despite the

identification of some virulence markers [Drevinek and

Mahenthiralingam, Clin Microbiol Infect 16:821-830],

many details of Bcc pathogenicity remain to be clarified.

To initiate infection, B. cenocepacia must be able to col-

onize the respiratory epithelium by binding to extracellu-

lar-matrix components. This step, although not fully

characterized, is believed to be in part mediated by a

specific type of adhesins, named trimeric autotransporter

adhesins (TAAs). TAAs are outer membrane proteins

that have emerging as virulence factors in Gram-

negative bacteria. They play critical roles in bacterial

adherence, biofilm formation, serum resistance, and

invasion to host cells [Linke et al., Trends Microbiol

14:264-270, 2006]. TAAs are multi-domain proteins or-

ganized in a modular fashion with a membrane-

anchored C-terminal conserved domain that forms a

trimeric beta-barrel pore and permits, through the type V

secretion system, the translocation of a passenger do-

main (stalk and an N terminal head) to the bacterial cell

surface [Łyskowski et al., Adv Exp Med Biol 715:143-

158, 2011].

Model of a typical TAA showing the domain architecture with

the anchor, stalk and head.

3D models of a TAA membrane-anchor region; monomer (a),

trimer (b) and upper view of the trimeric structure (c).

We have identified in silico 7 TAA-encoding genes in the

genome of B. cenocepacia J2315 [2]. Among those, 3

TAA genes (BCAM0219, BCAM0223 and BCAM0224)

are located within a cluster (named TAA cluster) on

chromosome 2 of B. cenocepacia J2315 [Mil-Homens et

al., Microbiology 156:1084-1096].

In this research project, we are interested in defining the

roles of these TAAs during Bcc infection. We believe our

work has relevance with respect to provide insights on

mechanisms used by Bcc to adhere, invade and persist

in the airways of CF patients.

38

Main achievements:

The genetic organization and the transcriptional

organization of the TAA cluster were determined. Be-

yond the 3 TAA-encoding genes, the TAA cluster has 6

other genes respectively coding for 1 lipoprotein

(BCAM0220), 2 sensor histidine kinases (BCAM0218,

BCAM0227), and 3 response-regulators (BCAM0221,

BCAM0222, and BCAM0228)

Genomic organization and operon mapping of the TAA

gene cluster

Quantitative real-time PCR analysis indicates that

the gene expression profiles occurred preferentially for

cells grown under high osmolarity, oxygen limited condi-

tions and oxidative stress. Furthermore, BCAM0224

gene, was exclusively detected in the epidemic ET-12

lineage and may serve as a valuable new addition to the

field of Bcc diagnostics.

We have constructed TAA mutants, and tested

their ability to adhere to extra-cellular matrix compo-

nents, to form biofilm or to hemagglutinate blood cells.

We also analysed each mutant for resistance to comple-

ment killing and we have used the killing of Galleria

mellonella to study the role of the adhesin genes in viru-

lence.

We investigate the roles of the B. cenocepacia

TAAs in host cell adhesion/invasion, using human cystic

fibrosis (CF) and non-CF airway epithelial cell lines.

Altogether, our results demonstrate that the B. cenoce-

pacia TAAs are multifunctional proteins participating in

cellular adhesion, serum resistance and virulence.

Adhesion of B. cenocepacia K56-2 (red) to 16HBE14o- (non-

CF) and CFBE41o- (CF) epithelial cell lines. The green color

reflects the eukaryotic cells plasma membranes while the blue

color represents the nucleus.

We are currently extending our studies to identify the

nature of the host cell surface receptors that interact with

the TAA proteins. Furthermore, we also intend to deter-

mine the molecular details of serum resistance mediated

by the TAAs. We believe that our studies may aid in the

development of treatments for Bcc infections in the lungs

of CF patients.

Funded project:

Virulence-associated Trimeric kAutotransporter Adhe-

sins from Burkholderia cenocepacia: functional, bio-

physical and structural characterization, PTDC/BIA-

MIC/118386/2010, PI: Arsénio M. Fialho

Selected publications:

[1] Mil-Homens D., Bernardes N., Fialho A.M. “The

antibacterial properties of docosahexaenoic omega-3

fatty acid against the Cystic Fibrosis multi-resistant path-

ogen Burkholderia cenocepacia” FEMS Microbiology

Letters 328:61-69, 2012

[2] Mil-Homens D., Fialho A.M. “Trimeric autotrans-

porter adhesins in members of the Burkholderia cepacia

complex: a multifunctional family of proteins implicated

in virulence” Frontiers in Cellular and Infection Micro-

biology doi: 10.3389/fcimb.2011.00013, 2011

39

20 11 Annual Report

Articles in International Peer-Reviewed

Journals

Abdulrehman D, Monteiro PT, Teixeira MC, Mira

NP, Lourenço AB, dos Santos SC, Cabrito TR,

Francisco A, Madeira SC, Santos R, Oliveira, AL,

Sá-Correia I, Freitas AT, YEASTRACT: Providing a

programmatic access to curated transcriptional regu-

latory associations in Saccharomyces cerevisiae

through a web services interface. Nucleic Acids

Research, 39, D136-140, 2011.

Cabrito TR, Teixeira MC, Singh A, Prasad R, Sá-

Correia I, The yeast ABC transporter Pdr18 (ORF

YNR070w) controls plasma membrane sterol compo-

sition, playing a role in multidrug resistance. Bio-

chemical Journal, 2011, 440, 195-202, 2011.

Coutinho CP, de Carvalho CCCR, Madeira A, Pinto

-de-Oliveira A, Sá-Correia I, Burkholderia cenocepa-

cia phenotypic clonal variation during three and a half

years of residence in the lungs of a cystic fibrosis

patient. Infection and Immunity, 79: 2950-60, 2011.

Coutinho CP, dos Santos SC, Madeira A, Mira NP,

Moreira AS, Sá-Correia I, Long-term colonization of

the cystic fybrosis lung by Burkholderia cepacia com-

plex bacteria: epidemiology, clonal variation and ge-

nome-wide expression alterations, Frontiers in Cel-

lular and Infection Microbiology, 1:12, doi: 10.3389/

fcimb.2011.00012, 2011.

dos Santos SC, Sá-Correia I, A genome-wide

screen identifies yeast genes required for protection

against or enhanced cytotoxicity of the antimalarial

drug quinine, Molecular Genetics and Genomics,

286(5-6), 333-326, 2011.

Ferreira AS, Silva IN, Oliveira VH, Cunha R,

Moreira LM, Insights into the role of extracellular pol-

ysaccharides in Burkholderia adaptation to different

environments. Frontiers in Cellular and Infection

Microbiology. 1:16, 1-9. doi: 10.3389/

fcimb.2011.00016, 2011.

Fialho AM, Chakrabarty AM, Law-medicine interfac-

ing: patenting of human genes and mutations, Recent

Patents on DNA Gene Sequence, 5, 81-85, 2011.

Gil FN, Gonçalves AC, Jacinto MJ, Becker JD,

Viegas CA, Transcriptional profiling in Saccharomy-

ces cerevisiae relevant for predicting alachlor mecha-

nisms of toxicity, Environmental Toxicology and

Chemistry, 30, 2506-2518, 2011.

Gonçalves JP, Francisco AP, Mira NP, Teixeira

MC, Sá-Correia I, Oliveira AL, Madeira SC, TFRank:

Network-based prioritization of regulatory associa-

tions underlying transcriptional responses, Bioinfor-

matics, 27, 3149-57, 2011.

Lourenço AB, Ascenso JR, Sá-Correia I, Metabolic

insights into the yeast response to propionic acid

based on high resolution 1H NMR spectroscopy.

Metabolomics, 7, 457-468, 2011.

Madeira A, Santos PM, Coutinho CP, Pinto-de-

Oliveira A, Sá-Correia I, Quantitative proteomics (2-

D-DIGE) reveals molecular strategies employed by

Burkholderia cenocepacia to adapt to the airways of

cystic fibrosis patients under antimicrobial therapy.

PROTEOMICS, 11, 1313-1328, 2011.

Mil-Homens D, Fialho AM, Trimeric autotransporter

adhesins in members of the Burkholderia cepacia

complex: a multifunctional family of proteins implicat-

ed in virulence, Frontiers in Cellular and Infection

Microbiology 1, article 13, 2011.

Publications

40

Mendes S, Farinha A, Ramos CG, Leitão JH, Vie-

gas CA, Martins LO, Synergistic action of

azoreductase and laccase leads to maximal decol-

ourization and detoxification of model dye-containing

wastewaters, Bioresource Technology, 102, 9852-

9859, 2011.

Mira NP, Henriques SF, Keller G, Matos R, Ar-

raiano C, Teixeira MC, Winge DR Sá-Correia I,

Identification of a DNA-binding site for the transcrip-

tion factor Haa1, required for Saccharomyces cere-

visiae response to acetic acid stress, Nucleic Acids

Research, 16, 6896-907, 2011.

Mira NP, Madeira A, Moreira AS, Coutinho CP, Sá

-Correia I, Genomic expression analysis reveals

strategies of Burkholderia cenocepacia to adapt to

the airways of cystic fibrosis patients and antimicro-

bial therapy, PLOS One, 6:12, e28831, doi:10.1371/

journal.pone.0028831, 2011.

Monteiro PT, Dias PJ, Ropers D, Oliveira AL, Sá-

Correia I, Teixeira MC, Freitas AT, Qualitative

modeling and formal verification of the FLR1 gene

mancozeb response in Saccharomyces cerevisiae,

IET Systems Biology, 5, 308-316, 2011.

Nadais H, Barbosa ML, Ramos CG, Grilo AM,

Sousa SA, Capela I, Arroja L, Leitão JH, Enhanc-

ing wastewater degradation and biogas production

by intermittent operation of UASB reactors. Energy

36, 2164-2168, 2011.

Pereira FB, Guimarães PMR, Gomes DG, Mira

NP, Teixeira MC, Sá-Correia I, Domingues L,

Identification of candidate genes for yeast engineer-

ing to improve bioethanol production in Very-High-

Gravity and lignocellulosic biomass industrial fer-

mentations, Biotechnology for Biofuels, 4, 57,

2011.

Ramos CG, Sousa SA, Grilo AM, Feliciano JR,

Leitão JH, The second RNA chaperone Hfq2, is

also required for survival to stress and the full viru-

lence of Burkholderia cenocepacia J2315. Journal

of Bacteriology, 193(7): 1515-1526, 2011.

Rocha J, Popescu AO, Borges P, Mil-Homens D,

Moreira LM, Sá-Correia I, Fialho AM, Frazão C,

Burkholderia cepacia UDP-glucose dehydrogenase

(BceC) structure and role of the Tyr10 in the final

hydrolysis of UGDs thioester intermediate, Journal

of Bacteriology, 193, 3978-3987, 2011.

Silva IN, Ferreira AS, Becker JD, Zlosnik JE,

Speert DP, He J, Mil-Homens D, Moreira LM, Mu-

coid morphotype variation of Burkholderia multi-

vorans during chronic cystic fibrosis lung infection is

correlated with changes in metabolism, motility, bio-

film formation and virulence, Microbiology,

157:3124-37, 2011.

Sousa SA, Ramos CG, Leitão JH, Burkholderia

cepacia complex: emerging multihost pathogens

equiped with a wide range of virulence factors and

determinants, International Journal of Microbiolo-

gy, Article ID: 607575, 2011.

Teixeira MC, Mira NP, Sá-Correia I, A genome-

wide perspective on the response and tolerance to

food-relevant stresses in Saccharomyces cerevisiae.

Current Opinion in Biotechnology, 22:150-156,

2011.

Teixeira MC, Cabrito TR, Hanif ZM, Vargas RC,

Tenreiro S, Sá-Correia I, Yeast response and toler-

ance to polyamine toxicity involving the drug:H+

antiporter Qdr3 and the transcription factors Yap1

and Gcn4. Microbiology, 157, 945-956, 2011.

Book Edition

Ramos, C.G., “Unveiling Novel Virulence Factors

from Burkholderia cepacia Complex: Exploitation of

the Caenorhabditis elegans infection model ”, Lam-

bert Academic Publishing, Saarbrücken, Germa-

ny, 2011 (ISBN: 978-3-8454-2283-1).

41

20 11 Annual Report

Book Chapters

T.R. Cabrito, E. Remy, M.C. Teixeira, P. Duque, I.

Sá-Correia, "Resistance to herbicides in the model

organisms Saccharomyces cerevisiae and Arabidop-

sis thaliana: the involvement of multidrug resistance

transporters", In: Herbicides and Environment

(Kortekamp A, Ed), INTECH, Vienna, Austria, 623-

640, 2011.

A.S. Ferreira, I.N. Silva, L.M. Moreira. (2011). Mech-

anisms controlling the expression of the exopolysac-

charide of Burkholderia and role in niche adaptation.

In Biotechnology of Biopolymers (M Elnashar Ed)

INTECH, Vienna, Austria (ISBN 978-953-307-179-4).

C.G. Ramos, A.M. Grilo, P.J.P. da Costa, H. Nada-

is, J.H. Leitão, "Extraction and Purification of DNA

from UASB Reactors Sludge Samples". In: DNA

Binding and DNA Extraction: Methods, Applica-

tions and Limitations (Chunxu Zhou and Xia Ling

Eds.). Nova Publishers. 2011. ISBN 978-1-61470-958

-9.

S.A. Sousa, J. Feliciano, J.H. Leitão, "Activated

Sugar Precursors: Biosynthetic Pathways and Biologi-

cal Roles of an Important Class of Intermediate Me-

tabolites in Bacteria". In: Biotechnology of Biopoly-

mers (Edited by Magdy M. Elnashar), Intech, 2011.

ISBN: 978-953-307-179-4.

Edition of Special Issues

Microbial Ecology and Global Health, Special Issue

of International Journal of Microbiology, 2011 (Jorge

H. Leitão, Guest Editor)

42

Patent applications

Fialho A.M., Bernardes N., Gonçalves J., Santos

A.C., “Compositions to treat HIV infections and meth-

ods thereof” 1126/MUM/2011, March 2011

Sá-Correia I., Cabrito T.R., Teixeira M.C., Remy E.,

Duque P., "Utilização de um gene que confere resis-

tência a xenobióticos em plantas", Patente de Inven-

ção Nacional, PT 105727, 2011.

Dissertations

Ph.D. Theses

Artur B. Lourenço, "Yeast responses and determi-

nants of resistance to propionic acid or ethanol at a

systems level: chemogenomic and metabolomic strat-

egies", PhD Thesis in Biotechnology, IST (Supervisor:

Isabel Sá-Correia), 2011.

Carla P. Coutinho, “Differentiation of clonal variants

of the Burkholderia cepacia complex isolated during

chronic respiratory infection in cystic fibrosis patients

with FTIR spectroscopy”, PhD in Pharmaceutical Sci-

ences-Analytical Chemistry, Faculty of Pharmacy-

University of Porto (Supervisor: João A. Lopes ; Co-

Supervisor: Isabel Sá-Correia), 2011.

Christian G. Ramos, "Role of sRNAs on the viru-

lence of the Burkholderia cepacia complex". PhD The-

sis in Biotechnology, IST (Supervisor: Jorge H. Leitão;

Co- Supervisor: Leonilde M. Moreira), 2011.

Tânia R. Cabrito, "Characterization of multidrug re-

sistance transporters in the model organisms Saccha-

romyces cerevisiae and Arabidopsis thaliana: estab-

lishing a link between physiological role and action in

chemical stress tolerance", PhD Thesis in Biotechnol-

ogy, IST (Supervisor: Isabel Sá-Correia; Co- Supervi-

sor: Miguel Cacho Teixeira), 2011.

M.Sc. Theses

Ana Sílvia Mendes Moreira, “Chronic pulmonary

colonization by Burkholderia cepacia complex bacte-

ria in Cystic Fibrosis patients: epidemiology, clonal

variation and gene expression analysis of sequential

isolates”, MSc in Applied Microbiology, Faculty of

Sciences of the University of Lisbon (Supervisor: Isa-

bel Sá-Correia), 2011.

Ana Teresa de Carvalho Estevens, "Expression,

purification and characterization of azurin derived

peptides as target sequences for P-cadherin overex-

pressing breast cancer cells"MSc in Biological Engi-

neering, Instituto Superior Técnico, Universidade Téc-

nica de Lisboa (Supervisor: Arsénio M. Fialho), 2011.

Andreia Filipa Tomás Marques, “Role of a tripartite

efflux pump in the symbiosis between Sinorhizobium

meliloti and leguminous plants”, MSc in Applied Mi-

crobiology, Faculdade de Ciências, Universidade de

Lisboa (Supervisor: Leonilde M. Moreira), 2011.

Joana Filipa Fernandes Guerreiro, “Molecular

mechanisms of adaptation and tolerance to acetic

acid in the food spoilage yeast Zygosaccharomyces

bailii”, MSc in Applied Microbiology, Faculty of Scienc-

es of the University of Lisbon (Supervisor: Isabel Sá-

Correia; Co-Supervisor: Nuno P. Mira), 2011.

Joana Nunes, "Functional analysis of the uncharac-

terized Candida glabrata drug:H + antiporters: role in

antifungal drug resistance of the antiporters CgAqr1

(ORF CAGL0J09944g) and CgTpo2/3 (ORF CA-

GL0I10384g)", MSc in Biomedical Engineering, IST

(Supervisor: Miguel C. Teixeira; co-supervisor: José

Melo-Cristino(FMUL)), 2011.

Joana Rita Rodrigues Feliciano, "Functional analy-

sis of the Burkholderia cepacia bceN gene encoding a

GDP-D-mannose dehydratase involved in cepacian

biosynthesis". MSc in Applied Microbiology, Fac-

uldade de Ciências da Universidade de Lisboa

(Supervisor: Jorge H. Leitão), 2011.

Paulo Jorge Gomes Pereira da Costa, “Pequenos

RNAs não codificantes em bactérias do complexo

Burkholderia cepacia: contribuição para a sua identifi-

cação e caracterização do seu papel na virulência".

MSc in Human Biology and Environment, Faculdade

de Ciências da Universidade de Lisboa (Supervisor:

Jorge H. Leitão), 2011.

43

20 11 Annual Report

Other Publications in International Jour-

nals

Editorials

Teplitski, M., Leitão, J.H., Sela, S., Editorial: Micro-

bial ecology comes of age during the global health

crisis. International Journal of Microbiology 2011.

Papers in Conference Proceedings

Bernardes, N, Ribeiro, AS, Seruca, R, Paredes, J,

Fialho, AM. “Bacterial protein azurin as a new candi-

date drug to treat untreatable breast cancers” In pro-

ceedings of “ENBENG 2011: 1st Portuguese Meeting

in Bioengineering”, Oeiras, Portugal, 2011, DOI

10.1109/ENBENG.2011.6026047.

Dias, PJ, Monteiro, PT, Costa, CP, Oliveira, AL,

Freitas, AT, Sá-Correia, I, Teixeira, MC. Using sys-

tems biology approaches to study a multidrug re-

sistance network. ENBENG 2011: 1st Portuguese

Meeting in Bioengineering, Oeiras, Portugal, 2011.

DOI: 10.1109/ENBENG.2011.6026073.

Ferreira, AS, Silva, IN, Becker, JD, Moreira, LM .

Role of tyrosine phosphorylation in the regulation of

Burkholderia cell physiology. Bioengineering

(ENBENG), 2011. 1st Portuguese Meeting, vol. no.1,

4 March pp.1-4.DOI:10.1109/ENBENG.2011.6026056

Grilo, AM, Ramos, CG, Sousa, SA, Arroja, LG,

Capela, IF, Leitão, JH, Nadais, HG. “Microbial popu-

lations shift as a toll for improving biogas production

in anaerobic reactors” In proceedings of the 12th Inter-

national Conference on Environmental Science and

Technology, Rhodes, Greece, 2011: A-625-629.

Madeira, A, Mira, NP, Santos, PM, Coutinho, CP,

Pinto-de-Oliveira, A, Moreira, AS, Roma-

Rodrigues, C, Sá-Correia, I. OMICS approaches to

reveal Burkholderia cenocepacia adaptive strategies

to long-term residence in the lungs of cystic fibrosis

patients under antibiotic therapy. ENBENG 2011: 1st

Portuguese Meeting in Bioengineering, Oeiras, Portu-

gal, 2011. DOI: 10.1109/ENBENG.2011.6026074.

Mil-Homens D, Fialho AM, “Identification and

characterization of novel multifunctional trimeric

autotransporter adhesins in the cystic fibrosis

pathogen Burkholderia cenocepacia” Gordon Re-

search Conference “Microbial Adhesion & Sig-

nal Transduction” Salve Regina University New-

port, RI July 24-29, 2011.

Ramos, CG, Sousa, SA, Grilo, AM, da Costa, PJP,

Feliciano, JR , Leitão, JH. A RNomics-based strate-

gy identifies regulatory small RNAs in Burkholderia

cepacia complex. ENBENG 2011: 1st Portuguese

Meeting in Bioengineering, Oeiras, Portugal, 2011.

DOI: 10.1109/ENBENG.2011.6026035.

dos Santos, SC, Teixeira, MC, Mira, NP, Cabrito,

44

TR , Palma, M, Lourenco, AB, Dias, PJ, Guerreiro,

J, Moreira, A, Henriques, S, Sá-Correia, I. Re-

sponse and resistance to drugs and chemical stress

in the yeast model: A genome-wide view. ENBENG

2011: 1st Portuguese Meeting in Bioengineering, Oei-

ras, Portugal, 2011. DOI: 10.1109/

ENBENG.2011.6026076.

dos Santos, SC, Palma, M, Tenreiro, S, Mira, NP,

Moreira, A, Sá-Correia, I. Identification of targets and

mechanisms of resistance to imatinib and quinine

using a molecular systems biology approach.

ENBENG 2011: 1st Portuguese Meeting in Bioengi-

neering, Oeiras, Portugal, 2011. DOI: 10.1109/

ENBENG.2011.6026077.

Sousa, SA, Ramos, CG, Feliciano, JR, Leitão, JH.

Identification and exploitation of Burkholderia cepacia

complex virulence factors as potential antimicrobial

targets. ENBENG 2011: 1st Portuguese Meeting in

Bioengineering, Oeiras, Portugal, 2011. DOI:

10.1109/ENBENG.2011.6026036.

Abstracts in international journals

Costa CP, Cabrito TR, Nunes J, Sá-Correia I,

Teixeira MC, "The putative Drug:H+ Antiporter

encoded by ORF CAGL0G08624g from Candida

glabrata confers multidrug resistance in yeast,

similarly to its S. cerevisiae homologue Qdr2."

Yeast 28(S1), S70, 2011).

Dias PJ, Monteiro PT, Oliveira AL, Freitas AT,

Sá-Correia I, Teixeira MC, "Insights into the tran-

scription regulatory network controlling the multi-

drug resistance gene FLR1: a systems biology

approach." Yeast 28(S1), S163, 2011).

Papers in National Journals

Fialho A.M. “Bactérias inimigas de tumores: novas

terapias para combater o cancro” Biologia e Socie-

dade, Revista da Ordem dos Biológos, 12: 14-18,

2011.

Online publications

T h e Y E A S T R A C T d a t a b a s e ( h t t p : / /

www.yeastract.com/) was updated and new queries

were made available providing a programmatic ac-

cess to curated transcriptional regulatory associations

in Saccharomyces cerevisiae through a web services

interface (authors at BSRG: M.C. Teixeira, N.P. Mira,

A.B. Lourenço, S.C. dos Santos, T.R. Cabrito, I. Sá-

Correia).

Deposition of Sequences in Databases

The DNA sequences of new allelic combinations were

added to the Burkholderia cepacia complex multilocus

sequence type (MLST) database (http://pubmlst.org/

bcc/) under the designation ST-614 (for 4 Burkhold-

eria cenocepacia isolates) and ST-668 (for 14

Burkholderia dolosa isolates), as well as two novel

alleles, gyrB-499 and trpB-344, both for B. dolosa,

and the MLST of 9 new B. contaminans isolates.

45

20 11 Annual Report

Invited Conferences

Sá-Correia I, Chemical stress defense mechanisms

in the Yeast model: molecular systems-biology ap-

proach, 14ª edição Portugaliae Genética (Model Or-

ganisms: Humans et al.), IPATIMUP, Porto, Portugal,

17,18 March 2011.

Teixeira MC, "Role and regulation of the Drug:H+

Antiporter family: from S. cerevisiae to C. glabrata",

keynote speaker at Microbiotec'11, Functional Ge-

nomics and Systems and Synthetic Biology session,

Braga, Portugal, 1-3 Dec 2011.

Oral Communications

Sousa SA, Ramos CG, Grilo AM, Feliciano JR,

Leitão JH. Burkholderia cenocepacia J2315 hfq2

gene plays a role on stress resistance and virulence.

4th Congress of European Microbiologists

FEMS2011, Geneve 25-30 June, Switzerland.

Dias PJ, Monteiro PT, Oliveira AL, Freitas AT, Sá-

Correia I, Teixeira MC, Insights into the transcription

regulatory network controlling the multidrug resistance

gene FLR1: a systems biology approach, 25th Interna-

tional Conference on Yeast Genetics and Molecular

Biology, 10-16 July 2011, Olsztyn-Kortowo, Poland.

Moreira LM. New molecular determinants of Sinorhi-

zobium-Medicago biological nitrogen fixation symbio-

sis. OCAST Workshop on Medicago transcriptomics,

data curation and data analysis. (2011) Samuel Rob-

erts Noble Foundation, Ardmore, USA

Ferreira AS, Silva IN, Becker JD, and Moreira LM.

Role of the tyrosine kinase BceF in Burkholderia

physiology. International Burkholderia cepacia work-

ing group (IBCWG). (2011) Prague, Czech Republic.

A17

Silva IN, Ferreira AS, Becker JD, and Moreira LM.

Adaptive developments of Burkholderia multivorans

clinical isolates during lung colonization – a tran-

scriptomic and phenotypic approach. International

Burkholderia cepacia working group (IBCWG). Pra-

gue, Czech Republic. A12

Communications in International Conferences

46

Poster Communications

Cabrito TR, Teixeira MC, Singh A, Prasad R, Sá-

Correia I. The yeast plasma membrane transporter

Pdr18 plays a role in plasma membrane sterol com-

position, conferring resistance to the herbicide 2,4-

D. 8th Lipidomics Meeting “Membranes and Bioac-

tive Lipids”, Lyon 26-28 October 2011.

Costa CP, Cabrito TR, Nunes J, Sá-Correia I,

Teixeira MC, The putative drug:H+ antiporter en-

coded by ORF CAGL0G08624g from Candida gla-

brata, confers multidrug resistance in yeast, similar-

ly to its S. cerevisiae homolog Qdr2 (Poster presen-

tation at the 25th International Conference on Yeast

Genetics and Molecular Biology, 10-16 July 2011,

Olsztyn-Kortowo, Poland).

dos Santos SC, Palma M, Tenreiro S, Moreira

AS, Sá-Correia I. Identification of targets and mech-

anisms of resistance to imatinib and quinine using a

molecular systems biology approach. 2011 CSHL

Systems Biology: Networks Meeting, New York 22-

26 May, United States.

Ferreira AS, Silva IN, Becker JD, and Moreira

LM. Role of BceF tyrosine kinase in Burkholderia

virulence and physiology. Meeting of the Irish Divi-

sion of Society for General Microbiology. Dublin,

Ireland.

Gil FN, Gonçalves AC, Becker JD, Viegas CA.

Gene expression profiling in the model yeast Sac-

charomyces cerevisiae to characterize and compare

the toxicity of six pesticides and identify biomarkers

of toxicity”, SETAC Europe 2011 – 21st Annual

Meeting of the Society of Environmental Toxicology

and Chemistry, May 2011, Milan, Italy.

Madeira A, dos Santos F, da Silva CL, Camafeita

E, Cabral JSM and Sá-Correia I. Molecular and

cellular networking underlying the ex-vivo expansion

of human mesenchymal stem cells (MSCs) revealed

by 2-DE based quantitative proteomics, The EMBO

Meeting, Vienna, 10-13 September, 2011.

47

20 11 Annual Report

Madeira A, Mira NP, Santos PM, Coutinho CP,

Pinto-de-Oliveira A, Moreira AS and Sá-Correia I.

Genome wide expression profiling reveals Burkhold-

eria cenocepacia adaptive strategies to long-term

colonization of the lungs of cystic fibrosis patients

under antibiotic therapy, The EMBO Meeting, Vien-

na, 10-13 September, 2011.

Monteiro PT, Dias PJ, Ropers D, Oliveira AL, Sá-

Correia I, Teixeira MC, Freitas AT, Computational

modeling and analysis of the Yeast FLR1 Regulato-

ry Network in Mancozeb-challenged Cells, 11th

International Conference on Systems Biology, Edin-

burgh, 10-15 October.

Santos MR, Marques AT, and Moreira LM. Role of

the Cpx two-component regulatory system and a

MFS transporter in the symbiosis between Sinorhi-

zobium meliloti and leguminous plants. 17th Interna-

tional Congress on Nitrogen Fixation. Fremantle,

Western Australia. P14

Sousa SA, Pereira L, Feliciano JR, Frazão C,

Leitão JH. Type II phosphomannose isomerase

BceA as a new target for the development of new

antimicrobials against Burkholderia cepacia com-

plex bacteria. 4th Congress of European Microbiolo-

gists FEMS 2011. Geneve 25-30 June, Switzerland.

48

Communications in National Conferences

Oral Communications

Bernardes N, Ribeiro AS, Mota B, Matos RG, Arraiano C,

Seruca R, Paredes J, Fialho AM Bacterial protein azurin as a

new candidate drug to treat P-cadherin overexpressing breast

cancer” MICROBIOTEC 2011, Braga 1-3 December 2011, Por-

tugal.

Guerreiro JF, Mira NP, Sá-Correia I, Adaptive response to

acetic acid in the highly resistant yeast species Zygosaccharo-

myces bailii, revealed by quantitative proteomics. MICROBI-

OTEC 2011, Braga 1-3 Dezembro 2011, Portugal.

Madeira A, dos Santos F, da Silva CL, Camafeita E, Cabral

JSM, Sá-Correia I, Molecular and cellular networking underlying

the ex-vivo expansion of human mesenchymal stem cells revea-

led by 2-DE based quantitative proteomics, MICROBIOTEC

2011, Braga 1-3 December 2011, Portugal.

Mira N, Henriques SF, Keller G, Teixeira MC, Matos RG,

Arraiano CM, Winge DR, Sá-Correia I, Haa1p-dependent regu-

latory network of the yeast response to acetic acid. MICROBIO-

TEC 2011, Braga 1-3 December 2011, Portugal.

Ramos CG, da Costa JP, Grilo AM, Becker J, Moreira LM,

Leitão JH. Hfq-like RNA chaperones and sRNAs: A new layer of

complexity in virulence regulation. MICROBIOTEC 2011, Braga

1-3 December 2011, Portugal.

Poster Communications

Cabrito TR, Teixeira MC, Singh A, Prasad R, Sá-Correia I. The

yeast plasma membrane transporter Pdr18 plays a role in plasma

membrane sterol composition, conferring multidrug resistance.

Microbiotec'11, Braga 1-3 December 2011.

Coutinho CP, Carvalho CCCR, Madeira A, Pinto-de-Oliveira A,

Sá-Correia I. Burkholderia cenocepacia clonal phenotypic variation

during long-term colonization of a cystic fibrosis patient lungs.

Microbiotec'11, Braga 1-3 December 2011.

Costa C, Cabrito TR, Sá-Correia I, Teixeira MC. Functional anal-

ysis of the uncharacterized Candida glabrata drug:H+ antiporter

CgQdr2 (ORF CAGL0G08624g): role in antifungal drug resistance.

Microbiotec'11, Braga 1-3 December 2011.

Cunha R, Silva IN, Ferreira AS, and Moreira LM. Biofilm for-

mation and protein-protein interaction within the exopolysaccharide

biosynthetic enzymes of Burkholderia cepacia complex isolates.

Jornadas de Engenharia Química e Biológica 2011, IST, Lisboa,

Portugal

Feliciano JR, Sousa SA, Pinheiro PF, Leitão JH. Functional

characterization of the BceN protein involved in GDP-D-rhamnose

biosynthesis by the Burkholderia cepacia complex. Microbiotec'11,

Braga 1-3 December 2011.

Fernanda M, Carvalho NN, Pinheiro PF, Leitão JH, Feliciano

JR. First insights into the biological activity of camphor type

species. XXII Encontro Nacional da SPQ - 100 Anos de Química

em Portugal. Braga 3-6 July 2011, Portugal

Gil FN, Gonçalves AC, Becker JD, Viegas CA. Gene expression

profiling in the model yeast Saccharomyces cerevisiae to charac-

terize and compare the toxicity of six pesticides and identify bi-

omarkers of toxicity”, Workshop Ciências e Engenharia do Ambi-

ente, IST-Ambiente, Lisboa, 22 Nov 2011

Gil FN, Gonçalves AC, Jacinto MJ, Becker JD, Viegas CA.

Genome-wide screening of molecular biomarkers in Saccharomy-

ces cerevisiae relevant for predicting alachlor toxicity”, MicroBi-

otec2011, Braga, Portugal, 1-3 Dez, 2011.

49

20 11 Annual Report

Grilo AM, Ramos CG, Sousa SA, Nadais H, Leitão JH. Molecular

characterization of microbial populations from UASB reactors treat-

ing dairy industry wastewaters. Microbiotec'11, Braga 1-3 Decem-

ber 2011.

Leitão JH, Capela I, Arroja L, Sousa SA, Ramos CG, Grilo AM,

Nadais H. Caracterização molecular de populações microbianas

de reactores anaeróbios a operar contínua ou intermitentemente

para o tratamento de efluentes. IST, Workshop Ciências e Engen-

haria do Ambiente, IST-Ambiente, Lisboa, 22 Nov 2011

Mendes S, Farinha A, Ramos CG, Leitão JH, Viegas CA, Mar-

tins LO. Synergistic action of azoreductase and laccase leads to

maximal decolourisation and detoxification of model dye-containing

wastewaters. Microbiotec'11, Braga 1-3 December 2011.

Mira NP, Madeira A, Santos P, Coutinho CP, Moreira AS, Pinto-

de-Oliveira A, Sá-Correia I.Transcriptomic and proteomic anal-

yses reveal Burkholderia cenocepacia adaptive strategies to long-

term colonization of the lungs of a cystic fibrosis patient under

antimicrobial therapy. Microbiotec'11, Braga 1-3 December 2011.

Moreira AS, Coutinho CP, Azevedo P, Lito L, Melo-Cristino J,

Sá-Correia I. Pulmonary co-infection by Burkholderia dolosa and

Burkholderia cenocepacia and clonal variation during long-term

colonization of a cystic fibrosis patient. Microbiotec'11, Braga 1-3

December 2011.

Ramos CG, Leitão JH. The cis-encoded h2cR small non-coding

RNA of Burkholderia cenocepacia J2315 is a post-transcriptional

negative regulator of hfq2. MICROBIOTEC 2011, Braga 1-3

Dezembro 2011, Portugal

Santos MR, Marques AT, and Moreira LM. New molecular deter-

minants of the symbiosis between Sinorhizobium meliloti and Medi-

cago sativa. Microbiotec'11, Braga 1-3 December 2011.

Silva IN, Ferreira AS, Tavares AC, and Moreira LM. Mucoid

morphotype variation of Burkholderia multivorans during chronic

persistence in the airways of cystic fibrosis patients. Microbi-

otec'11, Braga 1-3 December 2011.

Sousa SA, Pereira L, Feliciano JR, Frazão C, Leitão JH. Exploit-

ing the type II phosphomannose isomerase BceA as a target for

the development of new antimicrobials against Burkholderia cepa-

cia complex bacteria. Microbiotec'11, Braga 1-3 December 2011.

Viegas CA, Chelinho S, Moreira-Santos M, Mateus C, Costa

CP, Lima D, Ribeiro R, Fialho AM, Sousa JP. Evaluation of the

efficacy of a bioremediation tool based on soil bioaugmentation

with Pseudomonas sp. ADP in soils contaminated with herbicidal

commercial formulations containing s-triazines, Workshop Ciênci-

as e Engenharia do Ambiente, IST-Ambiente, Lisboa, 22 Nov

2011

50

Editorial Boards of International Scientific Jour-

nals

“FEMS Yeast Research”, “OMICS: a Journal of Integrative

Biology”, “International Journal of Microbiology”, “Journal of

Biomedicine and Biotechnology” (I. Sá-Correia).

“International Journal of Microbiology”, “The Open Microbi-

ology Journal”, “Bioengineered Bugs” (A.M. Fialho).

Guest Editors of International Journals

J.H. Leitão - Guest Editor of a Special issue of the

“International Journal of Microbiology” entitled “Microbial

Ecology and Global Health”

Organization of Scientific Events

9th Carbohydrate Bioengineering Meeting (CBM9), Lisbon, Portu-

gal, May 15-18, 2011 (I. Sá-Correia, Organizing Committee)

MICROBIOTEC 2011, Braga 1-3 Dezembro 2011, Portugal (I. Sá-

Correia and A.M. Fialho, Scientific Committee)

General and Committee Chairs or Committee

Memberships

Portuguese Society of Microbiology - (I. Sá-Correia, President ;

A.M. Fialho, 2nd secretary).

Council of the Federation of European Microbiological Societies

(FEMS) (I. Sá Correia, member)

Erasmus Mundus euSYSBIO Master’s Programme in Systems

Biology - I. Sá-Correia, coordinator at Instituto Superior Técnico

Evaluation panels

Portuguese Foundation for Science and Technology (FCT) individ-

ual research grants;- I. Sá-Correia.

European Research Council Advanced Grants - I. Sá-Correia.

Agency for the Assessment and accreditation of Higher Education

(A3ES) - I. Sá-Correia.

Awards

Honorable Mention of the UTL/Santander scientific awards 2011,

in the area of Biochemical Engineering and Biotechnology - Mi-

guel C. Teixeira

Honorable Mention of the UTL/Deloite scientific awards 2011, in

the area of Biochemical Engineering and Biotechnology - Nuno P.

Mira

Honorable Mention of the UTL/Deloite scientific awards 2011, in

the area of Biochemical Engineering and Biotechnology - Sílvia A.

Sousa.

Other Scientific Activities

eusYSBIO Winter School in Systems Biology 2012

EuSYSBIO: an ERASMUS MUNDUS Master Program in Systems Biology

The EuSysBio ERASMUS MUNDUS Master’s programme in Systems Biology (with KTH, Sweden,

and Aalto University, Finland) was coordinated at IST by Isabel Sá-Correia. Together with Miguel

C. Teixeira, they organized a two-week winter-school in Systems Biology (4-14 Jan 2011)

with open seminars held by Alfonso Valencia, CNIO, Spain, and Kiran Patil, EMBL, Germany.

51

20 11 Annual Report

BSRG Members

Faculty Staff Isabel Sá-Correia Arsénio Mendes Fialho Cristina Anjinho Viegas Jorge Humberto Leitão Leonilde Morais Moreira Miguel Cacho Teixeira

Post-doctoral Fellows Sílvia Andreia Sousa Nuno Mira Ana Sofia Ferreira Sandra dos Santos Margarida Palma Paulo Jorge Dias Carla Patrícia Coutinho

PhD Students Artur Bastos Lourenço Tânia R. Cabrito Christian G. Ramos Dalila Mil-Homens Catarina Rodrigues Andreia Madeira Mário Rui Santos Nuno Bernardes Inês Nunes Silva Fátima Gil Catarina Costa André M. Grilo

Research Assistants Alina Gonçalves Bruna Mota Filipa C. Roque Filipa Valada Sílvia Henriques Vera Lúcia da Silva

Master Students Ana Sílvia Moreira Andreia Marques Joana Nunes Joana F. Guerreiro Joana R. Feliciano Paulo JP da Costa Ana Almeida André Henriques Andreia Tavares Carla Alexandra Mateus Carla Pires Claudia S. Godinho Daniela Isidoro Eunice Penas Filipa G. Dias Filipe Silva Guida Camacho Kaur Alasoo Rita Maldonado Sílvia Matos Teresa Estevens Vítor H. Oliveira

Technical Assistant Mónica Rato

BSRG

Biological Sciences Research Group

Institute for Biotechnology and Bioengineering

Instituto Superior Técnico

Av. Rovisco Pais

1049-001 Lisbon

Portugal

http://ibb.pt/bsrg